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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4350-4360
Increases in Neutral, Mg2+-Dependent and Acidic,
Mg2+-Independent Sphingomyelinase Activities Precede
Commitment to Apoptosis and Are Not a Consequence of Caspase
3-Like Activity in Molt-4 Cells in Response to Thymidylate
Synthase Inhibition by GW1843
By
Ronald M. Laethem,
Yusuf A. Hannun,
Supriya Jayadev,
Connie J. Sexton,
Jay C. Strum,
Rebecca Sundseth, and
Gary K. Smith
From the International Science Development Group, the Departments of
Molecular Sciences, Functional Genetics, and Molecular Biochemistry,
Glaxo Wellcome Inc, Research Triangle Park; the Department of Medicine,
Duke University Medical Center, Durham; the National Institute of
Environmental Health Sciences, Research Triangle Park; and AndCare Inc,
Durham, NC.
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ABSTRACT |
Thymidylate synthase (TS) inhibition causes cell death, and this
enzyme is the target for the important chemotherapy regime 5-fluorouracil/leucovorin. GW1843 (1843U89) is a potent and specific folate analog TS inhibitor in clinical development. Because of the
importance of TS as a chemotherapy target, we are studying the
mechanism of TS inhibition-induced cell death by GW1843. Ceramide is a
regulatory lipid generated by the action of sphingomyelinase and is
believed to signal apoptosis. The role of the ceramide in apoptotic
signaling was studied in Molt-4 human T-cell leukemia cells undergoing
cell death after treatment with GW1843. In response to GW1843, Molt-4
cells undergo apoptosis with both acidic pH, Mg2+-independent sphingomyelinase (ASMase) and neutral
pH, Mg2+-dependent sphingomyelinase (NSMase) activities
elevated as early steps in the initiation of apoptosis before Molt-4
commitment to death. These activities lead to ceramide production with
kinetics consistent with a role as an effector molecule signaling the
initiation of apoptosis in Molt-4 cells. These changes were found to be
independent of caspase 3-like (CPP32/apopain) activity and DNA
degradation, but were not separable from membrane blebbing or cell
lysis in this cell line. In this report, kinetic evidence is provided
for a role of ceramide in initiating GW1843-induced cell death of Molt-4 T-cell leukemia cells.
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INTRODUCTION |
THYMIDYLATE SYNTHASE (TS)
catalyzes the synthesis of thymidylate from deoxyuridylate plus
5,10-methylenetetrahydrofolate and as such is critical for DNA
synthesis and cell growth. Inhibition of this enzyme produces cell
death.1-4 This enzyme is the target for the important
chemotherapy regime 5-fluorouracil/leucovorin. A number of new folate
analog TS inhibitors are now in clinical development.5-7
One of these agents is GW1843 (GW1843 has previously been reported in
the literature as 1843U89), a 3-methyl-substituted benzoquinazoline folate analog very specific for thymidylate
synthase.8,9
Because of the importance of TS as a chemotherapy target, we are
studying the mechanism of TS inhibition-induced cell death by GW1843.
In this context, we are interested in determining the means by which
the cell translates a TS inhibition event into commitment to cell
death. A more thorough understanding of these downstream elements may
facilitate development of novel agents and regimes to enhance important
TS-based therapies and to overcome natural and developed resistance
mechanisms. The goal of the current study was to investigate the
mechanism of possible apoptotic signaling pathways initiated in Molt-4
human T-cell leukemia cells in response to GW1843 therapy.
Apoptosis describes an orderly and regulated process by which cells
die. It has been known for some time that most cytotoxic anticancer
compounds kill by inducing apoptosis in susceptible cells.10 The effector molecules that couple the drug
stimulus to cell death are now beginning to be elucidated. One strong
candidate for such an effector molecule is ceramide. This lipid
molecule is the second messenger in the sphingomyelin (SM) signaling
pathway or "sphingomyelin cycle" as it has become
known.11 In this pathway, SM is metabolized by a
sphingomyelinase (SMase), yielding ceramide and phosphocholine.
Ceramide has been shown to be increased under a diverse array of
conditions that cause apoptosis12,13; however, it is not
known if ceramide is the actual intracellular mediator of the
extracellular death stimuli. The evidence that extracellularly applied
short chain synthetic analogs of ceramide and ceramide itself can mimic
the effects of various external apoptotic stimuli suggests that
ceramide is at least an integral component of the apoptotic signaling
pathway in a number of experimental systems.14 Two possible
effector molecules that are regulated by ceramide include the
ceramide-activated protein kinase15 and ceramide-activated
protein phosphatase.16 It is not known for certain what
role, if any, the substrates for the kinase or phosphatase may play in
apoptotic signaling.
Several different types of SMase isoforms have been described including
a membrane bound, Mg2+-dependent form (NSMase) active at
neutral pH,17 a lysosomal, Mg2+-independent
form (ASMase) maximally active at acidic pH,18 a secreted
Zn2+-dependent, acidic pH form produced from the same gene
as the lysosomal form,19 and a cytosolic,
Mg2+-independent, neutral pH form.20 The most
well characterized enzyme is ASMase, which is present in all cells and
is involved in the normal, lysosomal turnover of SM in the plasma
membrane. Patients with Niemann-Pick disease type A or B have either a
complete or partial loss of ASMase activity resulting in accumulation
of SM. NSMase has not been as well characterized and has not yet been
cloned. Both SMase enzymes have been reported to be activated in
response to tumor necrosis factor- (TNF-), and it has been suggested that ceramide generated by NSMase activates a
proline-directed serine/threonine protein kinase15 and
ceramide from ASMase activates NF- B.21 Interleukin-1
has also been reported to signal through a SMase
enzyme,22,23 which appears to be NSMase.24 It
has also been reported that cell death signaling through Fas/APO-1, a
receptor structurally related to p55 TNF-R, is mediated by the SM
signaling pathway.25 It is clear that the SM signaling
pathway is important for these processes, but it is not clear which
SMase enzyme is the most important for the particular cellular
responses induced by these cytokines. The determination of whether or
not there is a role for the SM signaling pathway as a general mediator of apoptotic stimuli will require further research.
We report here that in response to GW1843, both ASMase and NSMase
activities are elevated as early steps in the initiation of apoptosis
before Molt-4 commitment to death. These activities lead to ceramide
production with kinetics consistent with a role as an effector molecule
signaling the initiation of apoptosis in Molt-4 cells. These changes
were found to be independent of caspase 3-like (CPP32/apopain)
activity and DNA degradation, but were not separable from membrane
blebbing or cell lysis in this cell line. The implications for the
involvement of ceramide as a mediator of apoptosis in this cell line
are discussed.
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MATERIALS AND METHODS |
Reagents.
GW1843 was synthesized and used as described.26 Histopaque
1077 and 1119 were from Sigma (St Louis, MO). RPMI 1640 was from GIBCO (Grand Island, NY). Fetal calf serum (FCS)
was from JRH Biosciences (Lenexa, KS). Molecular weight markers were
from BioRad (Richmond, CA). Propidium iodide was from
Molecular Probes (Eugene, OR). Acetyl-asp-glu-val-asp-aldehyde
(Ac-DEVD-CHO), acetyl-asp-glu-val-asp-aminomethylcoumarin (Ac-DEVD-AMC), acetyl-tyr-val-ala-asp-aldehyde (Ac-YVAD-CHO), and
acetyl-tyr-val-ala-asp-aminomethylcoumarin (Ac-YVAD-AMC)
were from BACHEM Bioscience, Inc (King of Prussia, PA).
The caspase 3/interleukin-1--converting enzyme (ICE) assay kit was
a generous gift of Promega Corporation (Madison, WI) and was used
according to the manufacturer's instructions. Annexin-V coupled to
fluorescein (Annexin-V-Fluos) was from Boehringer Manheim Corporation
(Indianapolis, IN). All other reagents were supplied from standard
commercial sources.
Histopaque preparation of Molt-4 cell fractions.
Molt-4 cells were maintained in RPMI 1640 containing 10% FCS. Cells
were grown to log phase and seeded in 35 mL of RPMI 1640 with 10% FCS
in 75 cm2 flasks at 1 × 106/mL for
treatment with 8 nmol/L GW1843 and to 2.5 × 105/mL
for control studies. Cells from each flask were grown for 24 hours and
transferred to a 50-mL conical bottom centrifuge tube. A total of 10 mL
of a 1:1.8 mix of RPMI 1640:histopaque 1077 was underlayed beneath the
cells and 10 mL of histopaque 1119 was underlayed beneath that layer.
The tube was centrifuged at room temperature at 700g for 30 minutes. The cells were resolved into three fractions, the live
appearing cells form a band at the interface between the two histopaque
layers, the blebbed cells form a band at the interface between the
culture media and the 1:1.8 mix of RPMI 1640:histopaque 1077 and the
dead cells form a pellet. Cell fractions were harvested by aspirating
the media until approximately 1 cm remained above the top band of
cells. The cells were removed with a pasteur pipette, diluted 1:1 with media, and pelleted at 1,000g for 5 minutes. The media and
histopaque were aspirated and the cell pellet was washed with RPMI 1640 and resuspended in a suitable volume of media.
Analysis of DNA laddering by agarose gel electrophoresis.
DNA from Molt-4 cells was isolated by a modification of previously
published methods.27,28 Cells (1 × 106)
were pelleted by centrifugation at 1,500g and washed with
phosphate buffered saline. Cells were lysed with 100 µL of a solution
containing 50 mmol/L Tris-HCl pH 8.0, 10 mmol/L EDTA, 0.5% sodium
lauryl sarcosine, and 0.5 mg/mL proteinase K. After an overnight
incubation at 50°C, RNase A was added to 0.5 mg/mL and the sample
incubated at room temperature for 2 hours. Samples were extracted with
an equal volume (100 µL) of phenol:chloroform:isoamyl alcohol
(25:24:1) using Phase Lock Gel 1 Heavy extraction tubes (5 prime-3
prime, Boulder, CO) following the manufacturer's instructions.
Fragments of DNA from approximately 5 × 105 cells
were separated by electrophoresis on 1% agarose gels and visualized by
ultraviolet (UV)-transillumination.
Analysis of poly(ADP-ribose) polymerase (PARP) cleavage by Western
blotting.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
was performed as described29 with minor modification. Molt-4 cell aliquots (2 × 106 cells) were pelleted in
a microfuge tube and lysed in 50 µL of Laemlli sample
buffer.29 The samples were sonicated for 5 minutes in a
bath sonicator and incubated at 37°C for 1 hour. Samples were
electrophoresed in 7.5% polyacrylamide SDS gels at 200 V (constant
voltage) and blotted onto nitrocellulose using a Genie electrophoretic
blotter as instructed by the manufacturer (Idea Scientific Co,
Minneapolis, MN). Blots were blocked in 10% (wt/vol) nonfat dry milk
in Tris-buffered saline (TBS) overnight at room temperature. The blots
were incubated for 16 hours at room temperature in 5% (wt/vol) nonfat
dry milk in TBS containing 1:1,000 dilution of a monoclonal antibody
against PARP (kindly provided by Dr S.H. Kaufmann, Mayo Clinic,
Rochester, MN). Blots were washed and incubated with horse radish
peroxidase-conjugated secondary antibody for 1 hour at room
temperature. Resolved PARP was visualized using the ECL Western
blotting analysis system (Amersham, Arlington Heights, IL)
as instructed by the manufacturer.
Sphingomyelinase activity determination.
ASMase and NSMase activity was assayed using a trichloroacetic acid
(TCA) precipitation method in 96-well format. Assay buffer for NSMase
was 20 mmol/L Tris-HCl pH 7.4, 10 mmol/L EGTA, 5 mmol/L EDTA, and for
ASMase, it was 25 mmol/L NaOAc pH 5.0, 10 mmol/L EGTA, 5 mmol/L EDTA.
Both SMase activities were assayed in the presence and absence of 20 mmol/L MgCl2 to determine the Mg2+ dependence
of the SMase activity being assayed. Cells were treated and homogenized
at 2 × 107 cells/mL at 4°C in 20 mmol/L Tris-HCl
pH 7.4, 250 mmol/L sucrose, 10 mmol/L EGTA, 5 mmol/L EDTA, 0.3% Triton
X-100, 1 mmol/L benzamidine, 0.2 mmol/L
[4-(2-aminoethyl)benzenesulfonylfluoride, HCl] (AEBSF), 10 µmol/L
leupeptin, 10 µmol/L antipain, 1 µmol/L E-64, 1 µg/mL aprotinin,
and 1 µmol/L pepstatin A. Reactions (50 µL) contained 10 µL of
total Molt-4 homogenate (approximately 30 µg protein) in the
appropriate assay buffer and 1 µL of [14C]sphingomyelin
(New England Nuclear, Boston, MA) in methanol (54.5 Ci/mol, 7.4 µmol/L final concentration). Reactions were performed for
1 hour at 37°C and stopped with 5.6 µL of 10% (wt/vol) aqueous
fatty acid free bovine serum albumin (BSA) followed by 11.1 µL 50%
(wt/vol) aqueous TCA (reactions were found to be linear with time and
protein under these conditions). The 96-well plates were mixed well and
centrifuged at room temperature at 2,000g for 10 minutes. A
total of 40 µL of each supernatant was transferred to the
corresponding well of a Millipore (Bedford, MA) 96-well filtration plate (MAHV N45) and filtered under vacuum. Each well was
washed with 10 µL of ice cold 10% (wt/vol) aqueous TCA. The filtrate
was collected in a clear-bottom, opaque 96-well plate containing 200 µL of OptiPhase "Supermix" scintillant (Wallac, Gaithersburg,
MD). Plates were covered with mylar film, mixed until
homogenous, and counted on a Wallac Trilux µBeta 96-well plate
counter.
Caspase activity measurements.
ICE-like and caspase 3-like activity was analyzed in cell homogenates
prepared as for sphingomyelinase activity measurements. ICE-like
activity was determined against 50 µmol/L Ac-YVAD-AMC in the presence
or absence of the same concentration of Ac-YVAD-CHO. Caspase 3 activity
was determined against 50 µmol/L Ac-DEVD-AMC in the presence or
absence of the same concentration of Ac-DEVD-CHO. Assays were run
at 30°C in 100 µL total volume containing 32 µL of assay buffer
(from supplier), 53 µL of water, and 10 µL of 0.1 mol/L
dithiothreitol, and initiated with 5 µL of Molt-4 homogenate. Reaction progress was monitored in 96-well plates with a Perseptive Biosystems (Cambridge, MA) Cytofluor 4000 with excitation
and emission wavelengths of 360 and 460 nm, respectively. Activities were taken from the slope of the linear portion of the progress curve.
No activity was detectable when 50 µmol/L Ac-DEVD-CHO was included in
reactions where 50 µmol/L Ac-DEVD-AMC was used as the assay
substrate.
Lipid measurements.
Lipids were extracted from cells via the method of Bligh and
Dyer.30 Half of the extracted lipid was set aside for
phosphate determination,31 and the remaining half was used
in the diacylglycerol kinase assay as previously
described.32 Ceramide phosphate and phosphatidic acid spots
on thin-layer chromatography (TLC) plates were
quantitated using a Phosphorimager (Molecular Dynamics, Sunnyvale, CA).
External standards were run concomitantly to quantitate ceramide and
diacylglycerol levels and lipid levels were normalized to total lipid
phosphate.
Annexin-V binding.
Surface phosphatidylserine (PS) was measured by fluorescence microscopy
with the calcium-dependent PS binding protein Annexin-V coupled to
fluorescein isothiocyanate. Molt-4 cells at 1 × 106
cells/mL were exposed to 15 nmol/L GW1843 for 24 hours. This produced a
population of 75% live cells, 21% blebbed cells, and 4% lysed cells.
One million cells from this population were then washed twice with
Dulbecco's phosphate-buffered saline and resuspended in 100 µL of 10 mmol/L HEPES, 140 mmol/L NaCl, 5 mmol/L CaCl2. Annexin-V
binding was determined by fluorescence microscopy as fluorescein green
fluorescence.
Fluorescence-activated cell sorting (FACS) analysis.
Molt-4 cells were treated with 0, 8, and 16 nmol/L GW1843 and
timepoints taken at 24, 48, and 72 hours. Cells were fixed in 2 mL ice
cold 70% ethanol and stored at  20°C. Cells were pelleted at
1,500g for 5 minutes and resuspended at 1 × 106 cells/40 µL phosphate/citrate buffer containing 0.192 mol/L Na2HPO4 and 4 mmol/L citric acid pH 7.8. Cells were incubated for 30 minutes with gentle agitation and pelleted
as above. The supernatant was transferred to a fresh tube and the
pellets were resuspended in 1 mL propidium iodide (PI) buffer (3.4 mmol/L citric acid, 10 mmol/L NaCl, 50 µg/PI, 0.6% NP-40, and 37 µg/mL RNase). PI-stained DNA from the pellets was analyzed using a
Becton Dickinson (Franklin Lakes, NJ) FACSort instrument.
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RESULTS |
GW1843 induces cell death in Molt-4 cells.
The kinetics of Molt-4 cell death in response to 8 nmol/L GW1843
(IC50 for Molt-4 growth inhibition is 0.7 nmol/L) were
studied. Dead cells began to appear approximately 24 hours after
treatment. Three Molt-4 morphologic populations were observed: (1) live
cells which exclude trypan blue and appear normal; (2) blebbed cells which also exclude trypan blue and therefore apparently have intact membranes and have the nucleus marginated and compacted near the membrane; and (3) dead cells, which do not have intact membranes and
thus take up trypan blue.29 The time course for these
changes (Fig 1) indicates that at 24 hours
the Molt-4 cells are beginning to undergo death and by 48 hours 90% of
the cells are either blebbed or dead. At 18 hours, there was no
significant change in the number of cells in the blebbed or dead
fractions indicating that actual cell death begins to occur between 18 and 24 hours of GW1843 treatment.
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| Fig 1.
Time course for Molt-4 morphologic fractions formed after
treatment with 8 nmol/L GW1843. Molt-4 cells in log phase were treated with GW1843 and aliquots taken at the indicated times. Phase contrast microscopy was used to score cells as live ( ), blebbed ( ), or dead ( ) based on trypan blue exclusion. The graph represents the
average of three separate experiments (mean ± SEM).
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GW1843 induces apoptosis in Molt-4 cells as measured by morphology,
endonucleosomal DNA hydrolysis, specific PARP cleavage, and PS
externalization.
The GW1843-induced morphologic changes of Molt-4 cells observed with
phase contrast microscopy was also detected using transmission electron
microscopy.29 Morphologic hallmarks typical of apoptosis were clearly evident in electron micrographs including marginated chromatin and invaginated nuclei, micronuclei, and vacuolization of the
cytoplasm (data not shown).
Oligonucleosomal DNA laddering to approximately 180-bp fragments is
considered to be characteristic of apoptosis.33 The effect
of GW1843 treatment on Molt-4 cell DNA was examined using agarose gel
electrophoresis. A representative DNA analysis gel is shown in
Fig 2. Untreated control cells (lane 6)
contained approximately 7% dead cells as determined by trypan blue
exclusion. The DNA from these cells showed a faint ladder; however,
most of the DNA was intact. Cells treated for 48 hours with GW1843 contained 26% normal-appearing cells, 52% blebbed cells, and 22% dead cells. The DNA from these cells (lane 7) was mostly laddered with
only a small fraction migrating in the region of intact DNA. The
DNA from the treated cells clearly showed the approximately 180-bp
oligonucleosomal laddering indicating that the cells undergo apoptosis.
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| Fig 2.
Agarose gel electrophoresis of Molt-4 DNA. Molt-4 cells
were untreated or treated with 8 nmol/L GW1843 for 48 hours and
purified live, blebbed, and dead cell populations isolated on
histopaque gradients as described in Materials and Methods. Cellular
DNA was prepared and resolved by electrophoresis in 1% agarose gels gel as described in Materials and Methods. Lane 1, one kb ladder; lane
2, live fraction from untreated control cells after resolution by
histopaque gradients; lane 3, GW1843-treated, normal-appearing cells
after histopaque gradients (fraction contains approximately 6% dead
cells); lane 4, GW1843-treated, blebbed cells after gradients; lane 5, GW1843-treated, dead cells after gradients; lane 6, untreated cells
before gradients (fraction contains 93% normal cells and 7% dead
cells); lane 7, GW1843-treated cells before gradients (26% normal
cells, 52% blebbed, and 22% dead). The gel is from a representative
experiment from three separate determinations.
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The specific proteolytic cleavage of PARP, a nuclear protein involved
in DNA repair,34 is a marker of apoptosis.27,29 The effect of GW1843 on the integrity of PARP in the Molt-4 cells was
investigated using Western blot analysis
(Fig 3). At 24 hours of treatment,
degradation of the Mr 116,000 PARP to the
Mr 85,000 fragment was clearly shown, and only the
Mr 85,000 fragment was present at 48 hours. The
cleavage of PARP in GW1843-treated Molt-4 cells is further evidence
that GW1843 induces Molt-4 cells to die through an apoptotic process.
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| Fig 3.
Western blot of PARP proteolysis on treatment of Molt-4
cells with GW1843. Molt-4 cells in log-phase growth were treated with 8 nmol/L GW1843 and aliquots of 2 × 106 cells were taken at
the indicated times. Cells were prepared for electrophoresis,
electrophoresed on 7.5% polyacrylamide SDS gels,29 and
blotted onto nitrocellulose as described in Materials and Methods. The
blot is an x-ray film image of PARP protein detected with a horseradish
peroxidase-conjugated secondary antibody visualized with
chemiluminescent substrate (Amersham) blotted as described in Materials
and Methods. The Mr values were determined by
running prestained Mr markers (BioRad). Intact PARP
(upper band) is Mr 116,000 and the proteolytic
fragment is Mr 85,000. This blot is from a
representative experiment from four separate determinations.
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PS normally resides on the inner leaflet of the plasma membrane and is
externalized to the outer leaflet during apoptosis.35,36 Molt-4 cells were treated with 15 nmol/L GW1843 for 24 hours resulting in a cell population of 75% live cells, 21% blebbed cells, and 4%
lysed cells. Externalized PS was detected by treating with fluorescein
isothiocyanate (FITC)-labeled Annexin-V in the presence of calcium.
Figure 4 shows phase contrast (left) and
fluorescence microscopy (right) of these cells. In the phase contrast
photomicrograph, normal-appearing cells and blebbed cells with
apoptotic nuclei (arrows) were observed (in some fields, apoptotic,
lysed cells were also observed). However, only the blebbed and lysed
cells with apoptotic nuclei were found to bind Annexin-V, indicating that PS appearance on the outer leaflet corresponded with the apoptotic
morphology in the Molt-4 cells.
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| Fig 4.
Externalization of phosphatidylserine on 24 hours
treatment of Molt-4 cells with GW1843. Cells were collected, washed,
treated with FITC-Annexin-V, and viewed by phase contrast (left) or
fluorescein fluorescence (right) microscopy. Arrows indicate blebbed
cells. These photomicrographs are from a representative experiment from four separate determinations.
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Purification and characterization of Molt-4 morphologic populations
on histopaque gradients.
A histopaque centrifugation method was developed to separate the Molt-4
cell populations after treatment with GW1843 for 24 hours.
Table 1 indicates that at 24 hours, the
populations are highly enriched for the cell type of interest. The DNA
gel in Fig 2 shows the enrichment of the cell fractions after
histopaque gradient centrifugation. When untreated Molt-4 cells were
subjected to the centrifugation, the 7% dead cells in the population
from lane 6 were removed from the live cells as evidenced by the
absence of laddered DNA in the live fraction (lane 2). When the treated cells from lane 7 were purified on the gradients, the cell populations were resolved. The DNA from the live appearing cells (lane 3) was
largely intact; this cell population still contained 6% dead cells (as
determined by trypan blue staining) and the modest amount of laddered
DNA was consistent with this. The DNA from the blebbed and lysed cell
fractions (lanes 4 and 5, respectively) was completely laddered
indicative of cells having undergone apoptosis.
The viability of the three histopaque Molt-4 cell fractions was
investigated by determining their outgrowth potential. We have noted
previously that these cells are difficult to clone in agar, especially
after chemotherapy29; thus, viability was determined in
liquid culture. Cell fractions generated from histopaque centrifugation
after 48 hours treatment with GW1843 were placed back into culture
(0.05 × 106 cells/mL) and examined for outgrowth. In
standard media, control cells underwent five doublings in 96 hours
(doubling time is usually 21 hours under our conditions), while the
live cells underwent less than one doubling, presumably because of
persistent thymidylate synthase inhibition by GW1843
(Table 2). However, when 20 µmol/L thymidine was included in the media to bypass the TS
block,9 the live cell fraction proceeded through two
doublings in 48 hours and 3.9 doublings in 96 hours, one doubling less
than untreated cells, indicating that the cells in this fraction were
viable. The blebbed cells did not grow in culture with or without added thymidine (Table 2) indicating that they are committed to die.
GW1843-induced ceramide and SMase changes in Molt-4 cells during
apoptosis.
ASMase or NSMase have been implicated as components of the apoptotic
signaling cascade,37,38 although data on the true kinetic
competence of these enzymes in this signaling pathway is limited. The
kinetics of NSMase, ASMase, and ceramide changes during GW1843-induced
cell death are shown in Fig 5. Both ASMase and NSMase were elevated approximately fivefold over control after 18 hours of GW1843 treatment (Fig 5), a time when there was no reproducible increase in the number of blebbed or dead cells in the
population (Fig 1). The time course for ASMase activation was slightly
delayed compared with that for NSMase. At 18 hours, NSMase activity
peaked at 5.4-fold over control, whereas ASMase was 5.3-fold over
control, approximately 50% of its activity at 48 hours. By 48 hours,
NSMase activity had declined to 3.5-fold over control, but the ASMase
activity was further elevated to 10-fold over control in parallel with
the accumulation of dead cells.
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| Fig 5.
Time course of ceramide production and acidic pH,
Mg2+-independent and neutral pH,
Mg2+-dependent sphingomyelinase (ASMase and NSMase,
respectively) activation in total populations of Molt-4 cells treated
with 8 nmol/L GW1843. Log-phase Molt-4 cells were treated with 8 nmol/L GW1843, aliquots taken at the indicated times, and assayed for ASMase
activity (), NSMase activity (), and ceramide levels () as described in Materials and Methods. The graph represents the
average of four separate experiments (mean ± SEM).
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Ceramide is produced from the action of SMase on SM and has been
reported to be a regulatory molecule for apoptosis.11 We used diacylglycerol (DAG) kinase to assay ceramide levels in
GW1843-treated Molt-4 cells.32 Ceramide levels were
modestly elevated at 18 to 24 hours (2.2-fold over control, Fig 5). At
longer times, ceramide increases paralleled ASMase activity and
continued to increase to 48 hours, at which time ASMase was elevated
10-fold over control and ceramide levels were 7.7-fold over control.
The increases in ceramide are not due to an autocrine action of
TNF-39 in response to GW1843, as we were unable to
detect any increase in TNF- using a commercially available
enzyme-linked immunosorbent assay (ELISA) kit (data not shown). In
contrast to the large changes in ceramide, we found no reproducible
change in the level of DAG at any time point (data not shown).
Increases in SMase activity and ceramide levels in purified
populations of Molt-4 cells undergoing apoptosis.
To provide additional insight on the relationship of the SMases and
ceramide to GW1843-induced cell death, we determined their levels in
the resolved histopaque cell subpopulations. Molt-4 cells were treated
for 24 hours with 8 nmol/L GW1843, fractionated on histopaque, and the
fractions assayed for both ASMase and NSMase activity, as well as
ceramide levels. Interestingly, both ASMase and NSMase activities were
dramatically elevated in all three cell types, including the
normal-appearing cells with outgrowth potential
(Fig 6). Despite the elevated SMase
activity in all three cell types, the ceramide levels in the live cell
fraction were unchanged (1.3-fold over control). Ceramide levels were
substantially increased over control in the blebbed and dead cell
fractions, 3.8- and 2.4-fold, respectively; thus, ceramide was only
elevated in cells that were committed to apoptosis. These results, as
with the kinetics, support an initiation role for the SMases with
commitment to apoptosis occurring with ceramide accumulation.
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| Fig 6.
Ceramide, DAG, and acidic pH,
Mg2+-independent and neutral pH,
Mg2+-dependent sphingomyelinase (ASMase and
NSMase, respectively) activity in purified populations of
Molt-4 cells treated with GW1843. Log-phase Molt-4 cells were treated
with 8 nmol/L GW1843 for 24 hours, cell fractions were resolved on
histopaque step gradients, and assayed for ASMase activity (open),
NSMase activity (closed), ceramide levels (cross-hatched), and DAG
(shaded) as described in Materials and Methods. Control activity of
NSMase (mean ± SEM) was 3.3 ± 0.48 pmol/h/106 cells,
control activity of ASMase was 3.1 ± 0.18 pmol/h/106
cells and control levels of ceramide and DAG were 4.0 ± 1.8 and 29.2 ± 7.0 pmol/nmol lipid phosphate, respectively. The graph represents
the average of four separate experiments (mean ± SEM).
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Inhibition of caspase 3 does not affect activation of SMases or
accumulation of ceramide by GW1843.
To exclude the possibility that the activation of SMase and the
corresponding increase in ceramide is a consequence of apoptosis rather
than a signal for this process, we sought to inhibit apoptosis in the
Molt-4 cells in the presence of GW1843 and determine if increases in
ceramide still occurred. Caspase 3 is activated late in apoptosis to
cleave PARP and other proteins40 and can be inhibited in
vitro and in cells with Ac-DEVD-CHO.41 Others have shown
that inhibition of caspase 3 with this compound blocks apoptotic PARP
cleavage and DNA degradation.42 Changes in caspase 3-like and ICE-like caspase activity in response to GW1843 are shown in
Fig 7. Treatment with GW1843 alone resulted
in a maximal 35-fold increase in caspase 3-like activity over the
first 48 hours of treatment (paralleling cell death), which declined at
72 hours. Inclusion of the caspase 3 inhibitor Ac-DEVD-CHO in the
enzyme assay completely blocked the activity confirming that this was caspase 3-like activity (data not shown).
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| Fig 7.
Caspase activation by GW1843 in the presence and absence
of Ac-DEVD-CHO. Cells were treated for the indicated times with 8 nmol/L GW1843 alone () or in combination with 50 µmol/L
Ac-DEVD-CHO (). Aliquots were collected and assayed for caspase
3-like activity with Ac-DEVD-AMC as the substrate (solid lines) or
ICE-like activity using Ac-YVAD-AMC as the substrate (dashed lines).
Assays were performed as described in Materials and Methods. The data
in these plots are from a representative experiment from three separate determinations.
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When cells were treated with 50 µmol/L Ac-DEVD-CHO plus GW1843,
caspase 3-like activity was completely suppressed. This concentration of Ac-DEVD-CHO was found to be nontoxic to cells for at least 72 hours
(data not shown). That caspase 3 was inhibited in vivo by 50 µmol/L
Ac-DEVD-CHO was demonstrated by the inhibition of GW1843-induced DNA oligonucleosomal laddering determined with agarose
gel electrophoresis (Fig 8, left) and the
reduced formation of cells with <2N DNA per cell determined with FACS
analysis (Fig 8, right). Thus, the observed apoptotic DNA degradation
was dependent on caspase 3-like activity consistent with our
observation of PARP degradation. By contrast, 50 µmol/L Ac-DEVD-CHO
had no effect on the increases in ASMase or NSMase activities or
ceramide levels observed on GW1843 treatment
(Fig 9A).
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| Fig 8.
Effect of the caspase 3 inhibitor Ac-DEVD-CHO on
GW1843-generated apoptotic DNA. (Left) Log-phase Molt-4 cells were
treated for 48 hours with GW1843 alone or in combination with 50 µmol/L Ac-DEVD-CHO. Cells were harvested, DNA fragments isolated, and resolved using agarose gel electrophoresis as described in Materials and Methods. Lanes A, B, and C were treated with 0, 8, and 16 nmol/L
GW1843, respectively. Lanes D, E, and F were treated with 0, 8, and 16 nmol/L GW1843, respectively, in the presence of 50 µmol/L
Ac-DEVD-CHO. This gel is from a representative experiment from three
separate determinations. (Right) Log-phase Molt-4 cells were treated
for 48 hours as described below and prepared for FACS analysis as
described in Materials and Methods. Cells were treated as follows: (A),
mock treated cells; (B), 50 µmol/L Ac-DEVD-CHO; (C), 8 nmol/L GW1843;
and (D), 8 nmol/L GW1843 with 50 µmol/L Ac-DEVD-CHO.
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| Fig 9.
Changes in Molt-4 cells after treatment with GW1843. (A)
Changes in acidic pH, Mg2+-independent sphingomyelinase
activity (ASMase), neutral pH, Mg2+-dependent
sphingomyelinase activity (NSMase), and ceramide. Log-phase Molt-4
cells were treated for the indicated times with 8 nmol/L GW1843 alone
(solid lines) or in combination with 50 µmol/L Ac-DEVD-CHO (dashed
lines). Cell aliquots were assayed for ASMase activity (), NSMase
activity (), or ceramide levels () as described in Materials and
Methods. (B) Quantitation of dead cells (both blebbed and dead
fractions). Log-phase Molt-4 cells were treated for the indicated times
with 8 nmol/L GW1843 alone () or in combination with 50 µmol/L
Ac-DEVD-CHO (). Cells aliquots assayed for the number of blebbed and
dead cells after trypan blue staining as described in Materials and
Methods. This data is from a representative experiment from three
separate determinations.
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ICE-like activity was also quantitated during GW1843-induced apoptosis
of Molt-4 cells (Fig 7). This activity was not detectable under basal
conditions, but was detected maximally at 24 hours after GW1843
treatment. We estimate the increase over control activity to be
approximately twofold and at this level, was at least 25-fold less than
the maximal activation of caspase 3-like activity.
Quite surprisingly, while Ac-DEVD-CHO blocked DNA degradation, it had
no effect on the morphologic changes that occur during GW1843-induced
Molt-4 cell death (blebs, lysis, or nuclear margination). This is shown
in Fig 9B where it can be seen that kinetics for blebbed and lysed cell
accumulation were equal with and without Ac-DEVD-CHO.
To determine whether C2-ceramide could mimic the action of
the GW1843-induced intracellular ceramide increases, Molt-4 cells were
treated with 20 µmol/L C2-ceramide. This treatment
resulted in cell death and fragmentation of DNA, confirming previous
reports that ceramide can activate
apoptosis.11,13,14,25,43-47 Representative data are shown
in Fig 10. Treatment of Molt-4 cells for
17 hours with 20 µmol/L C2-ceramide led to accumulation
of cells with less than 2N DNA content (Fig 10B), similar to results
shown in Fig 8 for GW1843. Incubation of cells with 50 µmol/L
Ac-DEVD-CHO alone was without effect (Fig 10C); however, incubation of
cells with both 50 µmol/L Ac-DEVD-CHO and 20 µmol/L
C2-ceramide completely prevented the ceramide-induced
degradation of DNA (Fig 10D), as shown for GW1843 in Fig 8.
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| Fig 10.
Effect of caspase 3 inhibition on apoptotic DNA
fragmentation in response to C2-ceramide. Log-phase Molt-4
cells were treated as follows: (A) 0.2% dimethyl sulfoxide (DMSO) for
17 hours; (B) 20 µmol/L C2-ceramide for 17 hours; (C) 50 µmol/L Ac-DEVD-CHO for 35 hours; (D) 18-hour preincubation with 50 µmol/L Ac-DEVD-CHO followed by 17 hours coincubation with 20 µmol/L
C2-ceramide. Cells were harvested and prepared for FACS
analysis as described in Materials and Methods. This data is a
representative experiment from three separate determinations.
|
|
As with GW1843, Ac-DEVD-CHO had no effect on
C2-ceramide-dependent plasma membrane breakdown. After 8 hours, 76% of the cells were viable and 24% were dead in the presence
of C2-ceramide alone, and 79% cells were viable and 21%
were dead in the presence of the combination of C2-ceramide
and Ac-DEVD-CHO. After 17 hours, 22% of the cells were viable and 78%
were dead in the presence of C2-ceramide alone and 28% of
the cells were viable and 72% were dead in the presence of the
combination. Thus, as with GW1843-induced membrane changes in Molt-4
cells, C2-ceramide-induced membrane changes were also
independent of caspase 3-like activity.
| |
DISCUSSION |
The goal of this study was to investigate the role of ceramide and the
SMases in the apoptosis initiated by GW1843, a highly specific TS
inhibitor. The current data provide strong, novel evidence for their
intimate role in the process at a phase temporally preceding caspase 3 activation. Induction of apoptosis by GW1843 in the Molt-4 cells was
demonstrated using a number of criteria including morphologic changes
characteristic of apoptosis determined with phase contrast and
transmission electron microscopy, DNA degradation to the characteristic
incremental approximately 180-bp ladder, caspase 3 activation,
proteolytic cleavage of PARP from the intact Mr
116,000 form to the Mr 85,000 fragment, and
flipping of PS from the inner to the outer leaflet of the plasma
membrane. All of these changes were found to accumulate coordinately as normal-appearing live cells became blebbed and lysed cells. Consistent with this, the blebbed cells were found to be committed to cell death,
as thymidine, which bypasses the GW1843 block of TS, was not able to
rescue them, but was able to rescue a large proportion of the
normal-appearing, live cells.
The data in Figs 5, 6, and 9A show that ceramide accumulated in
parallel with the accumulation of dead cells. Thus at 24 hours, the
ceramide level in the live cell population was 1.3-fold over control,
whereas in the blebbed population, it was 3.8-fold over control. The
kinetics of ceramide accumulation in the total population (Figs 5 and
9A) were also consistent with increased ceramide levels signaling
commitment to apoptosis. Thus, only modest ceramide elevations  twofold over control, were found before 24 hours, the time at which the
blebbed and lysed cells began to appear. After this time, ceramide
levels increased along with the number of blebbed and lysed cells to
approximately eightfold over control by 48 hours.
Activation of NSMase and ASMase clearly preceded commitment to
apoptosis as shown in Figs 5, 6, and 9A. Thus, these enzymes (and
caspase 3) showed significant elevations in enzymatic activity by 18 hours, when few cells were committed to apoptosis. Indeed, NSMase was
fully activated by 18 hours. Activation of ASMase and caspase 3 lagged
behind NSMase; activity of both of these enzymes increased through 48 hours. Because NSMase activation and increased ceramide generation
precede caspase 3 activation, it is feasible that NSMase-derived
ceramide provides the caspase 3 activation signal. That activation of
the SMases preceded commitment was also shown using the histopaque
purified cell fractions. Thus, both enzymes were elevated about 10-fold
in the purified live cell fraction, and no further increase was
observed in either activity in the apoptosis-committed blebbed cells.
PS has been shown to externalize during apoptosis.35,36 We
showed here that this is also observed during GW1843-induced cell
death, and the PS externalization observed here occurred after the
morphologic change from the normal-appearing viable cells to blebbed
cells (Fig 4). An important aspect of our observed activation of the
SMases in the viable cell fraction, before conversion to the blebbed
cells, is that SMase activation cannot, therefore, be a result of the
PS flip. Thus, SMase activation is upstream of the PS flip. Whether the
PS flip is a result of SMase activity cannot be resolved from our data,
however.
Activation of the SMases before commitment, as shown from the activity
in the live cell fraction, is a necessary condition for a role of these
enzymes in initiating commitment to apoptosis. To further elucidate the
sequence of events, Molt-4 cells were treated with a combination of
GW1843 to initiate the apoptotic process, plus Ac-DEVD-CHO to block
cellular caspase 3-like activity. In these experiments, we asked
whether this caspase 3 inhibition would prevent SMase activation and
ceramide accumulation. Neither SMase nor ceramide levels were affected
by the addition of the caspase 3 inhibitor that completely blocked
caspase 3-like activity and DNA degradation (Figs 7 and 8). The
results clearly show that SMase activation is not dependent on prior
caspase 3 activation. Rather, the data show that SMase activation and
ceramide accumulation are either upstream of caspase 3 activation or on
parallel, independent paths. Consideration that ceramide itself can
lead to caspase 3 activation and/or DNA degradation as shown
here and elsewhere,11,13,14,25,43-47 strongly suggests that
the ceramide generated via SMase activity in response to GW1843 is
indeed upstream of caspase 3.
The observation that Ac-DEVD-CHO was able to block GW1843-induced DNA
degradation, but not bleb formation or lysis of Molt-4 cells, was
unexpected, and we do not yet completely understand its significance.
Nonetheless, it clearly shows that membrane events including SMase
activation, ceramide accumulation, and cell lysis can proceed in the
absence of caspase 3-like activity or DNA breakdown, which may be a
chemotherapeutic advantage by circumventing some cellular resistance
mechanisms. The mechanism by which apoptosis is initiated with a
membrane-generated signal in the absence of a DNA degradation signal
awaits further investigation. This observation is consistent with
another recent report on the effect of a caspase inhibitor on
apoptosis.48 These investigators observed that ZVAD-FMK
completely blocked cleavage of PARP and DNA on induced BAX expression.
This treatment, however, had no effect on the fall in mitochondrial
membrane potential, production of reactive oxygen, or membrane blebbing
induced by BAX, indicating that this latter set of changes was caspase
independent. Taken together, the data reported here and previously
indicate that molecules like BAX and GW1843 activate lethal membrane
events and DNA degradation likely in parallel. In the case of GW1843, ceramide may be involved in signaling because ceramide can be shown to
activate caspase 3-like activity,46,47 and, as we show
here, ceramide can produce cell lysis in the presence of blocked
caspase 3 activation and DNA degradation.
Which SMase is the relevant enzyme for the production of ceramide
involved in the membrane or DNA degradation events reported here is
unclear. While the NSMase was activated earlier in the time course of
GW1843 treatment, a role for the ASMase cannot be ruled out; indeed
activation of ASMase more closely paralleled accumulation of ceramide
and apoptotic cells. It is feasible that both enzymes are involved.
Others have reported that fibroblasts from ASMase-deficient
Niemann-Pick patients or some tissues from ASMase knockout mice that do
not express ASMase are resistant to apoptosis.49 Similarly,
it has been reported that loss of NSMase activity is associated with
resistance to apoptosis.50,51 Experiments with specific
inhibitors of each enzyme will be required to dissect the roles of the
individual SMases in the process of GW1843-induced cell death in Molt-4
cells.
An important, but nascent area of investigation, is the mechanism of
the SMase activation observed in the many conditions and cell types
studied. It is not known whether the increases in SMase activities
reported here and elsewhere are due to induction of enzyme synthesis or
to activation of preexisting enzyme. Because the time course for
activation of the SMases in our system is rather long, it is
conceivable that both processes could be at work. The activation of
NSMase in response to TNF- has been proposed to occur via indirect
coupling of the enzyme to p55 TNF-R through the coupling protein, FAN,
without the need for protein synthesis.21,38,52 Similarly,
the activation of ASMase in response to TNF- has been proposed to
occur via coupling to another region of the TNFR1 intracellular
domain.21 Whether, the apoptotic signal generated by GW1843
is transduced via a cell surface receptor is unknown, but we found no
detectable Molt-4 synthesis of TNF- in response to GW1843 treatment,
and Molt-4 cells do not respond to an anti-Fas-induced apoptotic
signal53 (C.J.S., unpublished observations).
In summary, we have shown that GW1843 induces apoptosis in Molt-4
cells. Prior to commitment to apoptosis, both NSMase and ASMase are
activated with a parallel increase in ceramide levels. Caspase 3 inhibition, which completely blocks DNA degradation, does not influence
the activation of either SMase or the accumulation of ceramide.
Further, the caspase inhibitor does not influence membrane breakdown
due to either GW1843 or ceramide in this cell line. The results support
a role for the SMase(s) and ceramide in the initiation of apoptosis at
both the membrane and nuclear level. The order of events in
GW1843-induced apoptosis of Molt-4 cells consistent with our
observations is shown in Fig 11. The main
processes include inhibition of TS, followed by loss of thymidine triphosphate and accumulation of deoxyadenosine
triphosphate (dATP);54 activation of NSMase
is followed by activation of ASMase resulting in the generation of
ceramide from SM. There are two distinct pathways at this point, in one
activation of caspase 3 (perhaps by a signal initiating from ceramide)
leads to PARP degradation and DNA laddering. In the other pathway,
ceramide increases lead to plasma membrane blebbing and lysis.
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| Fig 11.
Schematic diagram representing the major processes
involved in GW1843-induced apoptosis of Molt-4 cells. GW1843 inhibits
the production of thymidine monophosphate (TMP), which is
essential to DNA replication, resulting in accumulation of dATP. An
uncharacterized signal causes the activation of both acidic pH,
Mg2+-independent sphingomyelinase activity (ASMase) and
neutral pH, Mg2+-dependent sphingomyelinase activity
(NSMase). An increase in intracellular ceramide levels as a consequence
of the increased SMase activity causes plasma membrane breakdown and
may also activate caspases. The caspase pathway is sensitive to CPP32
inhibition and is characterized by events recognized as hallmarks of
apoptosis such as PARP cleavage and DNA laddering.
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|
| |
FOOTNOTES |
Submitted September 24, 1997;
accepted February 1, 1998.
Address reprint requests to Ronald M. Laethem, PhD, Glaxo
Wellcome, Inc, 5 Moore Dr, Research Triangle Park, NC 27709.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract