©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell Cycle Arrest Induced by an Inhibitor of Glucosylceramide Synthase
CORRELATION WITH CYCLIN-DEPENDENT KINASES (*)

(Received for publication, August 15, 1994; and in revised form, November 8, 1994)

C. S. Sheela Rani (1) Akira Abe (1) Yan Chang (1) Nitsa Rosenzweig (1) Alan R. Saltiel (2) Norman S. Radin (1) James A. Shayman (1)(§)

From the  (1)Nephrology Division, Department of Internal Medicine, University of Michigan-MSRB II, Ann Arbor, Michigan 48109-0676 and the (2)Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

In an attempt to define the basis for sphingolipid regulation of cell proliferation, we studied the effects of glucosylceramide (GlcCer) synthase inhibition by threo1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on NIH 3T3 cells overexpressing insulin-like growth factor-1 (IGF-1) receptors. PDMP treatment resulted in a time-dependent decrease in GlcCer levels and an increase in cellular ceramide levels. PDMP abolished serum and IGF-1-stimulated cell proliferation, as measured by a reduction in [^3H]thymidine incorporation, protein, and DNA levels. However it did not affect IGF-1-mediated early signaling events, including receptor tyrosine kinase, MAP kinase, and phosphatidylinositol 3-kinase activities. Two-color flow cytometry with propidium iodide and 5-bromo-2`-deoxyuridine monophosphate labeling revealed an arrest of the cell cycle at G(1)/S and G(2)/M transitions in an asynchronous population of cells. These changes were time dependent, with maximal effects seen by 12-24 h. Removal of PDMP from the cell medium resulted in reversal of the cell cycle changes, with cells re-entering the S phase. The cell cycle arrest at the G(1)/S and G(2)/M transitions was confirmed in cells synchronized by pretreatment with nocodazole, aphidicolin, or hydroxyurea, and released from blockade in the presence of PDMP. A decrease in the activities of two cyclin-dependent kinases, p34 kinase and cdk2 kinase, was observed with PDMP treatment. When cell ceramide levels were increased by N-acetylsphingosine, comparable changes in the cell cycle distribution were seen. However, sphingomyelinase treatment was without effect. Therefore, it appears that ceramide mediates in part the inhibitory effect of GlcCer synthase inhibition on IGF-1-induced cell proliferation in 3T3 cells. The rapid production of decreased cyclin-dependent kinase activities by PDMP suggests that one of the crucial sites of action of the inhibitor lies in this area.


INTRODUCTION

Sphingolipids have been identified as potentially important metabolites in the regulation of cell proliferation and differentiation. Sphingoid bases, which are the precursors and breakdown products of complex sphingolipids, can inhibit protein kinase C(1) , activate cellular kinases(2, 3) , and stimulate DNA synthesis and cell division in quiescent cultures of Swiss 3T3 cells(4) . Ceramides may be liberated as a result of agonist-stimulated sphingomyelin hydrolysis and are associated with activation of cellular kinases, phosphatases, and nuclear transcription events(5) . Ceramide or a related metabolite may mediate apoptosis(6) . Glycosphingolipids have been implicated in a variety of growth-related phenomena. They may regulate receptor tyrosine kinase activity(7) , phospholipases(8) , other kinases(9, 10) , and cell-cell and cell-matrix interactions(11) .

In recent years, attempts to understand specific cell functions of sphingolipids have been aided by the discovery of inhibitors of sphingolipid metabolism. These include inhibitors of 3-ketodihydrosphingosine synthase(12) , sphingoid base acyltransferase (13) , and beta-glucosidase(14) . The GlcCer (^1)synthase inhibitor, PDMP, has been particularly useful(15) . Treatment of cultured cells with the D-threo enantiomer results in the time-dependent depletion of all the glucosphingolipids. Secondary metabolic effects include the accumulation of ceramide and sphingosine. Inhibition of cell proliferation is observed in concert with these changes in sphingolipid levels(16) . In vivo studies demonstrate that PDMP treatment inhibits tumor growth and metastasis as well as renal hypertrophy associated with diabetes mellitus(17) .

The present study was initiated to investigate the possible role of sphingolipids in growth factor-stimulated cell proliferation. NIH 3T3 fibroblasts overexpressing receptors for insulin-like growth factor-1 (IGF-1) (18, 19) were used to examine PDMP effects on the growth factor signaling. We observed that treatment of 3T3 cells with the GlcCer synthase inhibitor abolished the proliferative response to either IGF-1 or serum. We also observed that PDMP did not affect early signal transduction events in response to serum or IGF-1, but it did block cell cycle progression in association with an inhibition of cell cycle-dependent kinases.


EXPERIMENTAL PROCEDURES

Materials

DL-Threo-PDMP was synthesized as described previously(20) . Receptor grade, human recombinant IGF-1 was from Baxter Scientific Products (McGaw Park, IL). N-Acetylsphingosine (C2-ceramide) and natural ceramide were from Matreya, Inc. (Pleasant Gap, PA), and phosphatidylinositol was from Avanti Polar Lipids, Inc. (Alabaster, AL). Sphingomyelinase (from Staphylococcus aureus, 120 units/mg protein), type III ceramide, bicinchoninic acid, 5-bromo-2`-deoxy-uridine (BrdU), propidium iodide, RNase A, nocodazole, hydroxyurea, and aphidicolin were from Sigma. Anti-BrdU-fluorescene isothiocyanate was from Becton-Dickinson Immunochemistry Systems (San Jose, CA). p34 kinase substrate peptide, histone H1, and Whatman P81 phosphocellulose discs were from Life Technologies, Inc. Protein G PLUS/Protein A-agarose suspension was from Oncogene Sci. (Uniondale, NY). Monoclonal antibodies to phosphotyrosine (PY20) and p34 kinase were from Transduction Laboratories (Lexington, KY). Anti-human cdk2 antibody (polyclonal rabbit antibody) was from Upstate Biotechnology Inc. (Lake Placid, NY). Mini-PROTEAN gel electrophoresis and Mini Trans-Blot transfer apparatus, Ready Gels, poly(vinylidine fluoride), and supported nitrocellulose membranes were from Bio-Rad. ECL Western blotting detection reagents, Hyperfilm-ECL, and [6-^3H]thymidine (28 Ci/mmol) were from Amersham Corp. [-P]ATP (25 Ci/mmol) was from ICN Radiochemicals (Irvine, CA). BA-S nitrocellulose membranes were from Schleicher & Schuell. The Prime-a-Gene labeling kit was from Promega Corp. (Madison, WI).

Cell Culture

NIH-3T3 mouse fibroblasts overexpressing IGF-1 receptors were used as described previously(18, 19) . These cells were routinely maintained in Dulbecco's modified Eagle's medium (with high glucose, glutamine, and sodium pyruvate, Life Technologies, Inc.), supplemented with 10% fetal bovine serum (Intergen, Purchase, NY), 100 units/ml penicillin, 100 µg/ml streptomycin, and 500 µg/ml Geneticin (Life Technologies, Inc.) under 5% CO(2) in a humidified incubator at 37 °C. Unless otherwise indicated, cells released by trypsin-EDTA treatment were plated at a density of 5 times 10^5 cells/100-mm Petri dishes in 10 ml of medium for 24-48 h. These cultures provided the asynchronous population of cells. Eighteen to 24 h prior to IGF-1 treatment, cells were growth arrested by replacement with the same medium, containing low serum (0.5%). IGF-1 was dissolved in the medium and added to cultures, while PDMP was dissolved in isopropyl alcohol, dried under nitrogen, and reconstituted in medium by sonication prior to addition to cultures. N-Acetylsphingosine was added in ethanol, the final concentration of ethanol being <0.1%.

[^3H]Thymidine Incorporation

The method of Kato et al.(19) was used. Confluent cells, grown in 24-well plates, were then grown in low serum medium for 24 h. They were incubated with IGF-1, with or without PDMP, for an additional 18 h. Cells were then incubated for 1 h with [6-^3H]thymidine (1 µCi/ml). The cells were rinsed twice with ice-cold PBS and precipitated with cold 5% trichloroacetic acid, then washed with cold ethanol, and solubilized with 0.5 N NaOH. Aliquots were assayed for protein utilizing the bicinchoninic acid reagent and for DNA radioactivity by scintillation counting.

IGF-1 Receptor Tyrosine Phosphorylation

Cells were grown to confluence and serum deprived for 24 h. The cultured cells were treated with or without 50 µM PDMP for 24 h. The cells were then incubated with or without IGF-1 for 5 or 30 min at 37 °C, washed three times with ice-cold PBS, and lysed with a buffer consisting of 1% Triton X-100 in 50 mM HEPES (pH 7.6), 100 mM NaCl, 10 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, and a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µM aprotinin, 10 µg/ml leupeptin). Tyrosine receptor phosphorylation was analyzed by immunoblotting with an anti-phosphotyrosine antibody, essentially as described by Kato et al.(19) .

Assay of MAP Kinase Activity

MAP kinase activity was assayed essentially as described by Miyasaka et al.(20) , except that myelin basic protein was used as the substrate.

Assay of Phosphatidylinositol 3-Kinase Activity

This assay was done essentially as described by Pang et al.(21) .

Cell Cycle Analysis by Flow Cytometry

The methods of Hoy et al.(22) and Kastan et al.(23) were employed to double-label cells with BrdU and propidium iodide. The cells were analyzed by flow cytometry at the University of Michigan Cancer Center's core facility.

Measurement of Cell Sphingolipid Levels

Cells were washed twice with 8 ml of cold PBS, scraped from Petri dishes with 2 ml of cold methanol, and transferred to screw cap tubes. Chloroform (2 ml) was added, and the mixture was vortexed and centrifuged at 2000 times g for 20 min. The pellet was reextracted with 4 ml of chloroform/methanol (1:1), and the supernatants were pooled. The protein content of the pellet was assayed by the method of Lowry et al.(24) . Chloroform (5 ml) and 4 ml of 0.9% NaCl were added to the pooled supernatants and the mixture vortexed and centrifuged at 800 times g. The lower layer was transferred to a glass tube, dried under nitrogen, and used for measuring ceramide and GlcCer levels.

Ceramide was measured by spotting lipid extracts and ceramide standards (0.2-2.0 µg) on thin layer chromatography plates and separating with chloroform/acetic acid (9:1). The ceramide spots were visualized by charring(25) , and the density of the spots was quantitated using a CCD video camera, connected to a Macintosh II computer utilizing NIH Image 1.49 software.

For GlcCer measurements lipid extracts were subjected to alkaline methanolysis by incubating each sample in 1 ml of chloroform and 0.5 ml of 0.21 N NaOH in methanol for 1 h at 37 °C. The solution was neutralized with 0.4 ml of 0.3 N acetic acid, vortexed, and centrifuged at 800 times g for 5 min. The lower layer was transferred to another glass tube and dried under nitrogen. The samples and GlcCer standards, dissolved in chloroform/methanol (2:1), were separated with borate-impregnated high performance thin layer chromatography plates developed with chloroform/methanol/water (63:24:4). The lipid spots were quantitated as above.

Assay of p34 Protein Kinase Activity Using a Peptide Substrate

The method of Marshak et al.(26) was employed using a specific 20-amino-acid p34 kinase peptide substrate. Following the experimental treatments, cells were washed with PBS and lysed with 1% Nonidet P-40 in a buffer consisting of 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM MgCl(2), 1 mM CaCl(2), 10% glycerol, 0.4 mM sodium orthovanadate, 10 mM EDTA, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml each of leupeptin and aprotinin). After 30 min on ice, the cell extracts were clarified by centrifuging 10 min at 14,500 times g at 4 °C. Protein was determined with the Bio-Rad DC reagent, and kinase activity was measured using 20-30 µg of lysate protein in a reaction mixture consisting of 1 mM peptide substrate in 50 mM Tris-HCl, pH 8.0, 10 mM MgCl(2), 1 mM dithiothreitol, 1 mM EGTA, and 100 µM ATP (1-2 µCi) in a total volume of 30 µl. After a 10-min incubation at 30 °C, 10% trichloroacetic acid was added and, after brief centrifugation, a 10-µl aliquot of the supernatant was applied to phosphocellulose paper discs. The disks were washed with 2 liters of 1% phosphoric acid and twice with water, and the radioactivity was measured by liquid scintillation. The appropriate blanks (counts/minute in the absence of enzyme and substrate peptide) were deducted from the total counts/minute, and the activity expressed as pmol of P/mg protein.

Assay of Histone Kinase Activity in Immune Complexes

The procedure of Rosenblatt et al.(27) was used. Cell lysates were prepared as described above for the assay of p34 kinase activity. Equal amounts of lysate protein (200 µg) were incubated with 2.5 µg of antibody for 1 h or overnight at 4 °C. A 30-µl aliquot of Protein A Plus/Protein G-agarose suspension was added and agitated for 1 h at 4 °C. The agarose beads were collected by centrifugation at 14,500 times g for 4 min at 4 °C. The beads were washed twice with 20 mM Tris-HCl, pH 7.5, containing 100 mM NaCl and 0.1% Triton X-100 and three times with kinase buffer consisting of 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 1 mM dithiothreitol. After removal of the last supernatant, the agarose pellet was resuspended in 30 µl of a reaction mixture consisting of 40 µg of histone H1, 25 µM ATP (2.5 µCi), and 10 mM MgCl(2) in the kinase buffer, and incubated for 15 min at room temperature. A 15-µl aliquot of the reaction mixture was spotted on phosphocellulose discs and the discs washed as described above. Another aliquot was used for measuring histone H1 phosphorylation, using SDS-polyacrylamide gel electrophoresis and autoradiography. For this, 15 µl of the reaction mix was boiled with Laemmli sample buffer and resolved on a 12% gel. The gel was stained with Coomassie Brilliant Blue and dried and the phosphorylated histone visualized by autoradiography.

Immunoblotting with Anti-cdk2 and Anti-p34 Kinase Antibodies

The lysates prepared as described for the kinase activity assay were mixed with 1 times Laemmli sample buffer, boiled for 5 min, and resolved on 12% gel by SDS-polyacrylamide gel electrophoresis. The proteins were electrophoretically transferred to poly(vinylidine fluoride) membranes as recommended by the manufacturer. The blots were first incubated with blocking buffer (1% bovine serum albumin in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20), and probed with anti-cdk2 or anti-cdc2 kinase antibodies. The immune complexes were detected using the ECL reagents or I-labeled goat anti-rabbit IgG (ICN) and quantitated by video densitometry as above.

Northern Blot Analyses of cdc2 mRNA

Northern analysis was performed as described previously(28) . Asynchronously growing 3T3 cells were treated for indicated periods with or without PDMP. The total cell RNA was isolated using the Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer's instructions. The purified RNA was dissolved in diethyl pyrocarbonate-treated water and quantitated by spectrometry as well as quantitative agarose gel electrophoresis. Equal amounts of total RNA were denatured in formaldehyde and formamide, fractionated on a 1% agarose formaldehyde gel, and transferred to BA-S nitrocellulose membranes. The RNA was then hybridized to an [alpha-P]dCTP randomly labeled 283-base pair cdc2 cDNA fragment. This probe was generated by reverse transcribed-PCR from 3T3 cells using primers 5`-GGC ACC ATA TTT GCT GAA CTA GC-3` and 3`-CGG TTT TAC CGT GAC TTA GTA GG-5`, subsequently cloned into Invitrogen PCR II vector by TA cloning and verified by DNA sequencing(29) . After overnight hybridization, the membranes were washed in high stringency conditions (twice with 2 times SSC, 0.1 times SDS at room temperature for 15 min each, twice with 0.2 times SSC, 0.1 times SDS at 55 °C for 30 min each), air dried, and the hybridization bands were visualized by autoradiography. As an internal control to assure equal RNA loading, the membranes were stripped and rehybridized with 139-base pair glyceraldehyde-3-phosphate dehydrogenase fragment as described above. This probe was generated by reverse transcribed-PCR from 3T3 cells using primers 5`-ATA TGA ATT CTC CAT GGT GGT GAA G-3` and 5`-ATG GGA AGC TTG TCA TCA-3`, also cloned into Invitrogen PCR II vector by TA cloning and verified by DNA sequencing(30) . All DNA fragments were labeled using the Prime-a-Gene kit. The hybridization bands on the autoradiograms were quantitated by video densitometry.


RESULTS

PDMP Inhibits Serum- and IGF-1-stimulated Growth

The inclusion in medium of 10% serum or 10 nM IGF-1 for 18 h to serum-deprived cells markedly stimulated DNA synthesis, as determined by [^3H]thymidine incorporation. The addition of 20 µM PDMP inhibited both the basal and growth factor-stimulated thymidine incorporation (Fig. 1). The effect of PDMP was dose dependent, with an observed IC of 15 µM for both IGF-1 and serum-stimulated proliferation. Some inhibition was seen with PDMP concentrations as low as 5 µM. PDMP also decreased cell protein and DNA content/dish, as previously reported in Madin-Darby canine kidney cells (16) .


Figure 1: Effect of PDMP on serum and IGF-1 stimulation of thymidine incorporation. NIH-3T3 cells grown to confluence were rendered quiescent by incubation in medium containing 0.5% serum. Cells were then incubated with no additions, with 10% serum or with 10 nM IGF-1, in the presence or absence of 20 µM PDMP for 18 h. [^3H]Thymidine (1 µCi/ml) was added for 1 h, the cells were washed, and the incorporated thymidine was measured as described under ``Experimental Procedures.'' The results are the mean ± S.E. of quadruplicate samples of a representative experiment.



The time dependence of the inhibition of cell proliferation by the GlcCer synthase inhibitor was assessed by delaying exposure of the cells to inhibitor following the addition of IGF-1. PDMP inhibited IGF-1-stimulated thymidine incorporation, and the extent of inhibition was attenuated as the duration of exposure to PDMP was shortened (Fig. 2). These data are consistent with the interpretation that the anti-proliferative effect of PDMP is the result of secondary metabolic effects and not due to the inhibitor itself.


Figure 2: Effect of delaying the time of PDMP addition following IGF-1 treatment on thymidine incorporation. Confluent cells grown to quiescence in serum-deprived medium were incubated for a total of 20 h with 10 nM IGF-1 with or without PDMP. PDMP (final concentration 20 µM) was added along with IGF-1 or at different times after IGF-1 addition. [^3H]Thymidine incorporation was determined as described under ``Experimental Procedures.'' Results, expressed as percent of IGF-1, are the mean ± S.E. of quadruplicate samples of a representative experiment.



PDMP Has No Effect on Early IGF-1-stimulated Signaling Events

Important steps in the cascade of early signaling events following IGF-1 binding are the activation of receptor tyrosine kinase, MAP kinase, and phosphatidylinositol 3-kinase (PI-3-kinase). Cells were growth arrested by serum deprivation, treated for 18 h with or without 50 µM PDMP, and then incubated with or without 10 nM IGF-1 for 5 or 30 min. Tyrosine phosphorylation of the IGF-1 receptor was examined by Western blotting with an anti-phosphotyrosine antibody. The stimulation by IGF-1 of tyrosine phosphorylation of the 97-kDa IGF-1 receptor beta subunit was unaffected by PDMP (Fig. 3). Other proteins demonstrating increased phosphorylation with IGF-1 stimulation were similarly unaffected.


Figure 3: IGF-1 receptor phosphorylation in cells treated with or without PDMP. Confluent cells grown to quiescence in serum-deficient medium were treated for 18 h with or without 50 µM PDMP. The cells were then incubated with 10 nM IGF-1 for 5 or 30 min, following which they were lysed and equal amounts of lysate protein were loaded on 7.5% SDS-polyacrylamide gels. The proteins containing phosphotyrosine were detected by Western immunoblotting as described under ``Experimental Procedures.'' The results are representative of four experiments. Lane 1, control untreated cells; lane 2, control cells incubated with IGF-1 for 5 min; lane 3, PDMP-treated cells, incubated with IGF-1 for 5 min; lane 4, control cells incubated with IGF-1 for 30 min; lane 5, PDMP-treated cells incubated with IGF-1 for 30 min. Arrow points to the 97-kDa phosphotyrosine-containing protein representing the IGF-1 receptor beta subunit.



Similarly, 24 h of pretreatment with 50 µM PDMP had no discernible effect on IGF-1 stimulation of MAP kinase (Fig. 4A) and PI-3-kinase (Fig. 4B) activities. These results clearly indicated that these initial events in growth factor action were not altered by PDMP pretreatment.


Figure 4: Effect of PDMP on IGF-1 stimulation of MAP kinase activity (A) and PI-3-kinase activity (B). Confluent cells grown to quiescence in low serum medium were treated with or without PDMP for 18 h. Cells were then incubated with or without IGF-1 for 5 min and lysed with detergent-containing lysis buffers, and the enzyme activities were determined as described under ``Experimental Procedures.'' The results, representative of two experiments each, are the mean ± S.E. of quadruplicate samples in each group.



PDMP Inhibits Cell Cycle Progression at G(1)/S and G(2)/M

The cell cycle progression of WT-3T3 cells exposed to the GlcCer synthase inhibitor was analyzed by flow cytometric analysis of DNA synthesis by BrdU incorporation and DNA content by propidium iodide-staining. Three distinct cell populations corresponding to G(0)/G(1), S, and G(2)/M phases were readily observed in an asynchronously growing population of untreated cells (Fig. 5A). Twenty-four h following exposure to 50 µM PDMP, marked and reproducible decreases in the percent of cells in the S phase and increases in the percent of cells in the G(2)/M phases of the cell cycle were observed (Fig. 5B). The cell cycle blockade by PDMP was reversed by incubating the cells in inhibitor-free culture medium for 24 h. This resulted in cell cycle redistribution with an increase in the percentage of S phase cells and a decrease in G(2)/M cells (Fig. 5C).


Figure 5: Flow cytometric analysis of cell cycle distribution in asynchronous cells treated with PDMP. Cells were plated at an initial density of 5 times 10^5/100-mm dishes in medium containing 10% fetal bovine serum for 24 h. The medium contained 0 or 50 µM PDMP for 24 h as shown. A, control cells; B, cells treated with PDMP for 24 h just before addition of BrdU; and C, cells treated with PDMP for 24 h were incubated for an additional 24 h in control medium. Cells from all treatments were fixed at the same time. The dot-plots show the simultaneous analysis of DNA synthesis by BrdU labeling to delineate the S phase and DNA content by propidium iodide labeling. Three discreet populations of cells representing G(0)/G(1) (2 N DNA with no BrdU incorporation (1), S-phase (variable DNA content showing BrdU incorporation) (2), and G(2)/M (4 N DNA content with no BrdU labeling) (3) are seen. The quantitation of cells in the three areas is given in the adjoining table.



The time dependence of the PDMP effect on cell cycle arrest in asynchronously growing cells was studied. A time-dependent increase in the percentage of cells in G(2)/M with a concomitant decrease in cells in the S phase was observed (Fig. 6A). These changes occurred in a parallel manner consistent with a block at both G(1)/S and G(2)/M. PDMP treatment for as little as 2 h resulted in significant changes in the distribution of cells. The peak effect was observed by 12 h with the percentage of cells in the G(2)/M phases increasing by >40% and the percentage of cells in the S phase decreasing by >50%.


Figure 6: Time course of PDMP effects on an asynchronous population of cells. The cells were treated without or with 50 µM PDMP for the indicated periods. All treatment groups were incubated simultaneously with BrdU and processed for flow cytometry. The results are the mean ± S.E. from three or four experiments, except for the groups of control and 24-h PDMP-treated cells, which had 11 experiments each. Results are expressed as percent of untreated control cells in each of the cell cycle stages. All the values are significantly different from control at p < 0.05 by unpaired t test. A, percent of cells in each cell cycle stage. B, relative concentrations of ceramide and GlcCer in cells treated as in the above experiment. Ceramide and GlcCer levels in control cells were 1.6 ± 0.15 and 1.52 ± 0.04 µg/mg protein, respectively. Results, expressed as percent of control, are the means from two experiments each. The fraction of cells in G(0)/G(1), G(2)/M, and S phases under control conditions were 85, 8.2, and 6.6%, respectively. This did not vary significantly in cells control cells at 48 h.



Cellular levels of ceramide and GlcCer were measured in parallel cultures treated similarly. A representative thin layer chromatography plate showing the separation of these lipids is shown in Fig. 7. The presence of N-acetylsphingosine can be seen in cells treated with this C2-ceramide in both solvent systems, demonstrating its cellular incorporation. Time-dependent increases in cell ceramide were observed, with levels increasing more than 3-fold by 24 h (Fig. 6B).


Figure 7: Thin layer chromatograms of lipids from NIH-3T3 WT21 cells. Panel A shows separation of ceramides from other lipids with chloroform/acetic acid (9:1). Panel B shows the separation of GlcCer with chloroform/methanol/water (63:24:4). Lanes A-F, ceramide and GlcCer standards, 0.2, 0.5, 0.75, 1.0, 1.5, and 2.0 µg. Lipid extracts from cells grown in low serum for 24 h and treated for 18 h as follows: lane 1, control; lane 2, IGF-1; lane 3, IGF-1 + PDMP; lane 4, IGF-1 for 18 h + PDMP for the first 6 h only; lane 5, IGF-1 + sphingomyelinase; lane 6, IGF-1 + acetylsphingosine. Arrows point to N-acetylsphingosine (N-Acsph). Lipid standards were comprised of type III ceramide (Sigma) and GlcCer obtained from Gaucher spleen.



GlcCer levels decreased by greater than 70% at 24 h. These changes in sphingolipid levels were consistent with the well-characterized ability of PDMP to inhibit GlcCer synthesis(15) . Cell protein/dish over the same time period decreased as well (not shown).

Cell Cycle Changes Are Reproduced by Short Chain Ceramides But Not by Sphingomyelinase Treatment

The apparent correlation between PDMP-induced cell cycle arrest and increased ceramide levels was examined further. Cells were treated with cell-permeable short chain ceramide, N-acetylsphingosine, or with sphingomyelinase. Both treatments significantly increased the levels of endogenous ceramides (Fig. 7) to levels comparable to those observed with PDMP treatment. Exposure of cells to 10 µMN-acetylsphingosine resulted in an increase in the fraction of cells in G(2)-M but did not decrease the fraction in S phase. However, sphingomyelinase failed to mimic the cell cycle effects of PDMP (Table 1).



The possible association between cell cycle block and increased ceramide levels was examined further in synchronized cells (Fig. 8, A and B). Cells were rendered quiescent by serum deprivation and then stimulated with 10 nM IGF-1. Treatment with IGF-1 for 18 h resulted in an almost 4-fold increase of cells in S phase, with a concomitant reduction in G(0)/G(1) phase, and a small, but significant increase in G(2)/M cells. Addition of PDMP with IGF-1 blocked the increase in S phase cells, the majority of cells being arrested in G(0)/G(1). Neither N-acetylsphingosine nor sphingomyelinase treatment inhibited entry of cells into the S phase. In contrast to the asynchronously growing cells, the G(2)/M population displayed no significant difference in the presence or absence of PDMP or N-acetylsphingosine.


Figure 8: Cell cycle distribution (A) and cellular sphingolipid levels (B) in serum-deprived cells treated with IGF-1 ± PDMP, C2-ceramide, or sphingomyelinase. Cells rendered quiescent by serum deprivation for 24 h were treated 18 h longer with 10 nM IGF-1 in the presence or absence of 25 µM PDMP, 10 µM acetylsphingosine, or 4 milliunits/ml sphingomyelinase (SMase). The results of the cell cycle study (mean ± S.E. of three to six experiments) are presented as percent of cells in the indicated cell cycle stages in each treatment group. The ceramide and GlcCer levels (mean ± S.D. of two experiments) are expressed as percent of untreated control, the control values being 2.17 ± 0.64 and 1.72 ± 0.5 µg/mg protein, respectively.



The effect of newly described inhibitors of GlcCer synthase on the cell cycle distribution was also determined. BML-130 is the DL-analog of threo-PDMP in which pyrrolidine and palmitic acid substitute for the morphiline and decanoic acid groups, respectively(31) . IV-231B is the D-enantiomer of an analog of BML-130 in which the benzene ring has been replaced by the alkenyl chain of sphingosine(32) . The latter compound inhibits GlcCer synthase, depleting cells of GlcCer, but does not result in ceramide accumulation(31) . Treatment with each inhibitor resulted in the accumulation of cells in G(2)/M; however, only PDMP treatment resulted in a decrease in the number of cells in the S phase (Table 2).



PDMP Effects on the Cell Cycle in Synchronized Cells

Several agents arrest growth at specific stages of cell cycle. Growth synchronization can be achieved following release from this blockade. Flow cytometric analysis was performed on cells treated with three cell cycle blockers (Fig. 9). A 16 h treatment with aphidicolin (4 µM) or hydroxyurea (10 mM) arrested cells in G(0)/G(1), with 85-90% cells in that stage (Fig. 9, panels B and C). Treatment with 2.5 µM nocodazole blocked cell cycle progression in G(2)/M, as shown by the high proportion (90%) of cells in that stage (Fig. 9, panel D).


Figure 9: Flow cytometric dot plots of asynchronous population of cells treated for 16 h with 4 µM aphidicolin (panel B), 10 mM hydroxyurea (panel C), or 2.5 µM nocodazole (panel D). Panel A shows control cells. Percentage of cells in G(0)/G(1), S and G(2)/M (areas marked 1-3, respectively) are shown in the adjoining table.



The cell cycle distribution was studied when cells were released from this blockade in the presence or absence of PDMP (Fig. 10). In each case release from blockade by control medium resulted in recovery of the S phase. Following blockade with aphidicolin and hydroxyurea, PDMP treatment resulted in an increase in cells at G(0)/G(1) and G(2)/M and a decrease in cells at the S phase. In contrast, PDMP treatment following release from mitotic arrest by nocodazole resulted in a block at G(1)/S with no change at G(2)/M.


Figure 10: Effect of PDMP on cell cycle distribution of cells synchronized by pretreatment with aphidicolin (APH, panel B), hydroxyurea (HU, panel C), or nocodazole (NOCO, panel D). Asynchronous population of cells were treated with or without (C, panel A) the above agents as mentioned in the legend for Fig. 10. After 16 h, the additions were removed by washing three times with medium, and the cells were continued to incubate for an additional 24 h in (1) control medium or (2) with 50 µM PDMP. The percentages of cells (mean ± S.D.) in G(0)/G(1), S and G(2)/M (areas marked 1-3, respectively) are shown in the adjoining table. The flow cytometric dot-plots are representative of two or three experiments.



PDMP Treatment Decreases p34 and cdk2 Kinase Activities

The effect of PDMP on two of the enzymes known to be involved in cell cycle regulation was studied. The activation of p34 kinase (cdc2), a serine-threonine protein kinase, is critical for progression of cells from interphase into mitosis or M phase(33) . Cdk2 is another member of the cell cycle-dependent kinases, which shares 60% homology with the cdc2 kinase, associates with cyclins A and E, and is maximal at G(1)/S transition(34) . Both cyclin A and cdk2 are associated with DNA in the initiation complex during its replication, and the retinoblastoma protein has been identified as a substrate for the cyclin-cdk2 complexes(35, 36) . In the initial experiments, the activity of p34 kinase in crude cell lysates was measured by phosphorylation of a synthetic peptide substrate that corresponds to a 21-amino-acid sequence surrounding the phosphorylation site of SV40 large T antigen, a native substrate of this enzyme(26) . PDMP treatment for 20 h caused a significant reduction in the activity of p34 kinase, as measured by this assay (Fig. 11A). Direct addition of 20 µM PDMP to the assay reaction mixture had no effect on kinase activity (data not shown). The reduction of cdc2 kinase activity could be seen within 1 h of PDMP addition, in contrast to the slower effect on ceramide level (Fig. 6B). Similar treatment with sphingomyelinase (Fig. 11A) had no effect on the activity of this enzyme, consistent with their lack of effect on cell cycle changes. When the lysates from the time course experiment were immunoblotted with specific cdc2 antibody (Fig. 11B), a reduction (37.5 ± 7.3%) was seen in a 34 kDa band corresponding to the p34 kinase 20 h after PDMP treatment (lanes 2 and 5) compared to control (lane 1), but not at 1 or 2 h after PDMP (lanes 3 and 4), nor after 20 h with sphingomyelinase (lane 6). These results show that the reduction in the cdc2 activity preceded the reduction in the protein.


Figure 11: p34 kinase activities and immunoblots. A, time course of p34kinase activity following treatment of asynchronous cells with PDMP (50 µM) or sphingomyelinase (4 milliunits/ml). Cdc2 kinase activity was measured in crude cell lysates using the peptide substrate as described under ``Experimental Procedures.'' Results expressed as percent of control are the mean ± S.E. of three to five experiments. The specific activity of cdc2 kinase in control lysate was 275 ± 17 pmol [P]/mg protein. *p < 0.05 versus control. B, immunoblot of p34kinase in cell lysates from the above experiment. The cell lysates were from the following treatments: lane 1, control; lanes 2 and 5, PDMP 20 h; lane 3, PDMP 1 h; lane 4, PDMP 2 h; and lane 6, sphingomyelinase (SMase), 20 h.



The activity of cdk2 and cdc2 kinases was determined by histone phosphorylation following immunoprecipitation with specific antibodies. PDMP treatment for 2 or 20 h of asynchronous cells caused a reduction in the activities of both cdc2 and cdk2 kinases (Fig. 12A). Similarly, treatment with hydroxyurea (which arrests cells in G(1)/S) also reduced the activity of both these enzymes. In contrast, treatment with nocodazole (which arrests cells in metaphase) resulted in an increase in cdc2 activity and a decrease in cdk2 activity (Fig. 12A).


Figure 12: Activities of cyclin-dependent kinases. A, asynchronous cells grown in serum-containing medium were treated for 2 or 20 h with 50 µM PDMP, or for 20 h with hydroxyurea (HU, 1 mM), or nocodazole (Noco, 2 µM). B, cells grown in serum-deprived medium for 24 h were treated with 10 nM IGF-1 in the presence or absence of 50 µM PDMP or 4 milliunits/ml sphingomyelinase for 18 h. Following the treatments, cells were lysed and the lysates subjected to immunoprecipitation using specific antibodies to p34 kinase (cdc2) and cdk2 kinase. The kinase activity of the immune complex was determined by histone H1 phosphorylation as described in the text. The results (percent of corresponding untreated control) represent the mean ± S.E. of three experiments, each sample assayed in duplicates.The specific activity of cdk2 kinase in control cells grown in medium with and without serum were 12.5 ± 1.3 and 5.8 ± 1.6 pmol [P]/mg protein, respectively. Similarly, the specific activity of cdc2 kinase in control cells grown in medium with and without serum were 2.8 ± 0.16 and 1.6 ± 0.3 pmol P/mg protein, respectively. *p < 0.05 versus corresponding control by unpaired t test. C, immunoblots of cdc2 kinase. Western blotting analysis with cdc2 antibody was done as described in the text. The lysates were from the same treatments as described in B: lane 1, control; lane 2, IGF-1; lane 3, IGF-1 + PDMP; and lane 4,: IGF-1 + sphingomyelinase.



The effect of PDMP on the cell cycle-dependent kinases was also studied in serum-deprived cells treated with IGF-1. The activity of both cell cycle-dependent kinases was increased almost 2-fold over control by IGF-1 treatment (Fig. 12B). Addition of PDMP along with IGF-1 significantly decreased these cell cycle-dependent kinase activities, while treatment with sphingomyelinase had no effect on the IGF-1-induced increase, consistent with the cell cycle data. Analysis of cdc2 by Western blotting (Fig. 12C) indicated that IGF-1 treatment for 18 h caused an induction of cdc2 kinase as shown by an increase in the 34 kDa band (lane 2, 2.6 ± 0.5-fold over control, lane 1). Inclusion of PDMP along with IGF-1 totally abolished this increase in cdc2 kinase (lane 3versuslane 2); sphingomyelinase addition along with IGF-1 had no effect on cdc2 (lane 4versuslane 2). Treatment of cells with 10 µMN-acetylsphingosine decreased the cdc2 kinase and cdk2 kinase activities to 76 and 88% of IGF-1-stimulated control values, respectively (data not shown). Similar analysis of cdk2 by Western blotting showed no change following the above treatments (data not shown).

PDMP Treatment Does Not Inhibit p34 Kinase mRNA Expression in Asynchronous Cells

Since there was a decrease in the cdc2 protein, as shown by Western blotting following 20 h of PDMP treatment, the effect of PDMP on p34 mRNA was studied. The cdc2 expression in asynchronous cells treated with PDMP was the same as in controls at all times examined (Fig. 13). PDMP also had no effect on glyceraldehyde-3-phosphate dehydrogenase expression.


Figure 13: Northern blot analysis of cdc2 mRNA expression. Asynchronous population of NIH-3T3 cells were incubated without (control) or with 50 µM PDMP for periods as indicated below. The total RNA was isolated and examined by Northern analysis as described under ``Experimental Procedures'' with cdc2 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fragments. Lane 1, PDMP, 1 h; lane 2, control, 1 h; lane 3, PDMP, 6 h; lane 4, control, 6 h; lane 5, PDMP, 12 h; lane 6, control, 12 h; lane 7, PDMP, 24 h; lane 8, control, 24 h.




DISCUSSION

Primary events following binding of various polypeptide growth factors, including IGF-1, to a family of receptors are the activation of protein kinases associated directly or indirectly with these receptors(37) . On binding, the kinases are activated to trigger a cascade of phosphorylation processes, culminating in DNA synthesis and cell proliferation.

In the present study we have investigated whether alterations in endogenous sphingolipids affect growth factor signaling via IGF-1. GlcCer synthase inhibition by PDMP blocked serum- or IGF-1-stimulated proliferative response as indicated by a decrease in [^3H]thymidine incorporation in 3T3 cells overexpressing the IGF-1 receptors. However, early IGF-1-stimulated signaling events, including receptor autophosphorylation, activation of MAP/basic protein kinase, and PI-3-kinase were unaffected. Thus, the antiproliferative site of action of PDMP must lie downstream to these early phosphorylation events.

The cell cycle analyses demonstrated that PDMP induced a specific cell cycle block at G(1)/S in both asynchronously growing cells and in cells synchronized in G(0) by serum deprivation. Release from cell cycle arrest with nocodazole also resulted in G(1)/S blockade. PDMP treatment also caused an additional block at G(2)/M.

The PDMP-induced inhibition of cell cycle progression was reversible; cells released from the block were able to re-enter the cycle, as shown by the recovery of S phase cells. The antiproliferative effects of PDMP on IGF-1-stimulated proliferation were time dependent; [^3H]thymidine incorporation was reduced even when the GlcCer synthase inhibitor was added several hours after IGF-1. However, PDMP was most effective in blocking the proliferative response when added simultaneously with the growth factor and became less effective when its addition was delayed. The effect of PDMP was therefore not due to direct inhibition of [^3H]thymidine uptake, but probably due to time-dependent changes in sphingolipid levels.

The growth inhibitory effects of PDMP were accompanied by a time-dependent increase in cell ceramide and a decrease in GlcCer levels. This is consistent with our earlier interpretation that blockade of GlcCer synthase is the cause of growth inhibition(15) . Cellular ceramide levels were also raised by sphingomyelinase; interestingly, C2-ceramide treatment also increased the endogenous ceramides, a finding previously observed in MDCK cells treated with a longer chain ceramide, octanoylsphingosine(38) . C2-ceramides are readily incorporated into cultured cells, and they have therefore been used extensively as a test of the biological activity of ceramide(39) . However, the secondary effects of short chain ceramides on the metabolism of endogenous sphingolipids, including glycosphingolipids and sphingomyelins, require that the use of these compounds be interpreted cautiously. A correlation was observed between PDMP effects on cell cycle changes and ceramide accumulation. Cell cycle inhibition was not seen when cell ceramide levels were increased by incubation with sphingomyelinase but was seen with C2-ceramide treatment.

Recently, a second messenger function for ceramide has been proposed. In this model the activation of a neutral sphingomyelinase by agonists generates ceramide(40) , which mediates the functional response to these agents (see (5) ). For example, in the leukemia cell line HL-60, ceramide has been suggested to mediate many of the effects of tumor necrosis factor-alpha, such as cell growth and differentiation(5) , programed cell death (or apoptosis)(6) , and activation of the nuclear factor kappaB(41) . However, other recent studies have shown that not all effects of tumor necrosis factor-alpha are mediated by ceramide(42, 43) . A recent study (44) has reported that treatment of T lymphocytes with agents such as interleukin-2 or phorbol ester plus ionomycin decreased the ceramide levels and increased [^3H]thymidine incorporation, while PDMP, which increased endogenous ceramide, inhibited [^3H]thymidine incorporation observed at 24 h or more. Furthermore, C2-ceramide and sphingosine analogs had less pronounced growth inhibitory effects, since a decrease in [^3H]thymidine incorporation could be seen only at 48, not in 24 h. Consistent with our observations, sphingomyelinase treatment, which also increased ceramide levels, did not inhibit T-cell growth. Based on these data, it was hypothesized that there may be functionally distinct pools of ceramide within the T cell.

Several studies have reported that the addition of some sphingolipids, such as ganglioside GM(3), results in inhibition of cell proliferation; conversely, metabolites of gangliosides may enhance proliferation. The effects of gangliosides in these studies have been correlated with the regulation of receptor kinase activities associated with the platelet-derived growth factor, epidermal growth factor(9, 10) , and insulin receptors(7) . PDMP treatment causes depletion of cell gangliosides(15) , at a speed depending on the rates of ganglioside turnover, and therefore could inhibit growth factor-stimulated cell proliferation by inhibition of receptor tyrosine kinase activity. PDMP has been reported to enhance tyrosine phosphorylation of T cells stimulated with interleukin-2(45) . Alternatively, ceramide has been identified as a potential mediator of EGF receptor phosphorylation at the threonine 669 site and therefore may regulate receptor kinase activity. However, the absence of observable changes in IGF-1 receptor phosphorylation, MAP kinase, or PI-3-kinase activities, suggests that ganglioside or ceramide-mediated effects on early signaling events cannot explain the antiproliferative effects of PDMP in 3T3 cells. Our preliminary results also suggest that sphingosine may not be the mediator of PDMP inhibition of cell proliferation, since addition of sphingosine (1-5 µM) by itself was growth stimulatory as indicated by an increase in [^3H]thymidine or BrdU incorporation. (^2)

Major progress has been made in recent years in our understanding of the regulation of cell cycle. Progression through cell cycle transition points in eukaryotic cells is controlled by the activation of cyclin-dependent kinases. The timing of activation of these kinases is regulated by association with regulatory subunits (cyclins), and by phosphorylation(34) . There now are well-documented examples of both positive and negative control mechanisms for these processes. In yeast, the control of G(1)/S and G(2)/M transitions is mediated by a single cell cycle-dependent kinase in association with different cyclins, while in mammalian cells, it is controlled by multiple cell cycle-dependent kinases in association with multiple cyclins; each of these complexes act at distinct cell cycle stages (for reviews, see Refs. 46, 47)

The effect of PDMP on two kinases, the p34 kinase associated with progression of cells into mitotic phase, and the cdk2 kinase which is activated at G(1)/S, was evaluated. The activity of p34 kinase was increased when the cells were arrested in mitosis by nocodazole treatment and markedly reduced when the cells were arrested at G(1)/S by hydroxyurea. Similar changes in cdc2 kinase activity were reported in Hela cells following treatment with nocodazole and hydroxyurea(48) . Both nocodazole and hydroxyurea treatments, however, reduced the cdk2 activity, probably reflecting a lack of cells in G(1)/S transition.

PDMP treatment of asynchronously growing cells caused a decrease in both the cdc2 and cdk2 kinase activities, consistent with a cell cycle blockade occurring at G(2)/M and G(1)/S transitions, respectively. Increasing ceramide levels with N-acetylsphingosine or other PDMP analogs partly replicated the G(2)/M block but not the G(1)/S block seen with PDMP. Since these inhibitors all block GlcCer formation to a comparable extent, the G(2)/M block is most likely due to ceramide or a ceramide catabolite and is unlikely to be due to cellular depletion of glycosphingolipids. Ceramide has recently been identified as a potential activator of protein phosphatase 2a. This phosphatase inhibits the transition from G(2) to mitosis by dephosphorylating cdc25 and p34(49) . It is tempting to speculate that the inhibitory effects of ceramide on G(2)/M transition are mediated by activation of this phosphatase.

In conclusion, the present study demonstrates that the GlcCer synthase inhibitor PDMP has specific and reversible effects on growth factor-stimulated cell growth in NIH-3T3 cells. PDMP treatment causes cell cycle arrest, in association with an inhibition of the cell cycle-dependent kinases. Early signaling events are not affected. Although the present study does not resolve the question of the mechanism for PDMP-induced growth inhibition, our results indicate that ceramide may contribute to this effect.


FOOTNOTES

(^2)
C. S. S. Rani, J. Choe, and J. A. Shayman, unpublished data.

*
This work was supported by National Institutes of Health Grants DK 39255 and DK41487 (to J. A. S.) and a Merit Review Award from the Department of Veterans Affairs (to J. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Established Investigator of the American Heart Association. To whom correspondence should be addressed: Nephrology Div., University of Michigan, Box 0676, 1560 MSRB II, Ann Arbor, MI, 48109. Tel.: 313-763-0987; Fax: 313-763-0982.

(^1)
The abbreviations used are: GlcCer, glucosylceramide; PDMP, threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; IGF-1, insulin-like growth factor-1; BrdU, 5-bromo-2`-deoxyuridine monophosphate; PBS, phosphate-buffered saline.


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