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(Received for publication, August 22, 1995; and in revised form, December 21, 1995) From the
Prior studies demonstrated that ceramide promotes apoptotic cell
death in the human myeloid leukemia cell lines HL-60 and U937 (Jarvis,
W. D., Kolesnick, R. N., Fornari, F. A., Jr., Traylor, R. S., Gewirtz,
D. A., and Grant, S.(1994) Proc. Natl. Acad. Sci. U. S. A. 91,
73-77), and that this lethal process is potently suppressed by
diglyceride (Jarvis, W. D., Fornari, F. A., Jr., Browning, J. L.,
Gewirtz, D. A., Kolesnick, R. N., and Grant, S.(1994) J. Biol.
Chem. 269, 31685-31692). The present findings document the
intrinsic ability of sphingoid bases to induce apoptosis in HL-60 and
U937 cells. Exposure to either sphingosine or sphinganine
(0.001-10 µM) for 6 h promoted apoptotic degradation
of genomic DNA as indicated by (a) electrophoretic resolution
of 50-kilobase pair DNA loop fragments and 0.2-1.2-kilobase pair
DNA fragment ladders on agarose gels, and (b)
spectrofluorophotometric determination of the formation and release of
double-stranded fragments and corresponding loss of integrity of bulk
DNA. DNA damage correlated directly with reduced cloning efficiency and
was associated with the appearance of apoptotic cytoarchitectural
traits. At sublethal concentrations ( Recent investigation has examined the participation of
sphingophospholipid- and glycerophospholipid-derived messengers in the
regulation of leukemic cell survival. We (1, 2) and
others (3) have demonstrated that increased intracellular
availability of ceramide induces programmed cell death or apoptosis in the human myeloid leukemia cell lines HL-60 and U937. Ceramide
interacts with at least two distinct intracellular target enzymes,
ceramide-activated protein kinase (4, 5, 6) and ceramide-activated protein
phosphatase (7, 8, 9) . A cytotoxic role for
ceramide-activated protein phosphatase and ceramide-activated protein
kinase in ceramide action has been inferred, although the relative
contributions of these enzymes to the initiation of apoptosis is
presently uncertain(10, 11) . A contrasting
cytoprotective function of diglyceride and, therefore, of one or more
isoforms of protein kinase C (PKC) ( In further support of a
central cytoprotective function for PKC, we have also described the
induction of apoptosis in HL-60 cells by pharmacological agents that
selectively inhibit activity of this isoenzyme family (e.g. calphostin C and chelerythrine)(12) . The importance of
sphingoid bases such as trans-4-sphingenine (sphingosine) and
4,5-dihydrosphingosine (sphinganine) as physiologically relevant
inhibitors of PKC is well established(13) . In addition, the
cytotoxic properties of sphingoid bases and other, more complex,
lysosphingolipids have been linked directly to inhibition of
PKC(14) . Both sphinganine and sphingosine have been shown to
reduce proliferative capacity and long term viability in HL-60
cells(15) . Ohta and co-workers recently examined the lethal
actions of sphingosine within the context of cellular maturation and
proposed that endogenous sphingosine mediates apoptotic cell death
following phorboid-induced terminal differentiation in HL-60
cells(16) . Apart from those studies, however, little
information is presently available concerning the apoptotic influences
of sphingoid bases in human leukemia cells. The present report
describes biochemical characterizations of direct and indirect
apoptotic properties of sphingoid bases in undifferentiated HL-60
cells. These findings demonstrate that acute exposure to sphingosine
and other sphingoid bases potently elicits apoptosis as assessed by
multiple criteria, including the induction of double-stranded DNA
damage, loss of clonogenic potential, and appearance of apoptotic
morphology. These results additionally reveal that co-exposure to
either sphingoid bases or selective pharmacological PKC inhibitors at
sublethal concentrations augments the apoptotic capacity of the lethal
lipid messenger ceramide. This interaction is mechanistically
consistent with our previous observations that, conversely,
ceramide-mediated cell death is suppressed by diglyceride and
pharmacological PKC activators(1, 2) . Thus, it
appears that the apoptotic response to ceramide is indirectly regulated
by the combined actions of sphingosine and diglyceride, which
respectively limit or extend the cytoprotective influence of PKC. Based
upon these observations, we propose that the reciprocal influences of
sphingoid bases and diglyceride on PKC coordinately modulate
ceramide-mediated apoptosis in human myeloid leukemia cells.
Figure 1:
Induction of apoptotic DNA degradation
by sphingoid bases. HL-60 cells were exposed to synthetic preparations
of sphingosine (So; 10 µM), sphinganine (Sa; 10 µM), or vehicle (Veh) for 6 h.
Apoptotic DNA fragments were resolved on agarose gels as described
under ``Experimental Procedures.'' Panel A,
resolution of loop (
Figure 2:
Quantification of sphingoid base-induced
DNA damage. HL-60 cells were exposed to synthetic preparations of
sphingosine (So; 10 µM), sphinganine (Sa; 10 µM), or vehicle (Veh) for 6 h.
DNA damage was then determined by quantitative spectrofluorophotometry
as described under ``Experimental Procedures.'' Panel A, formation (single-hatched bars) and release (double-hatched bars) of double-stranded DNA fragments; values
are expressed as nanograms of DNA/10
Figure 3:
Expression of apoptotic cytoarchitecture
in response to sphingoid bases. HL-60 cells were exposed to vehicle (Veh; panel A) sphingosine (So; 10
µM; panel B), sphinganine (Sa; 10
µM; panel C) for 6 h. Following fixation, cells
were stained with a modified Wright-Giemsa preparation and examined by
conventional light microscopy.
The apoptotic responses of
HL-60 cells to sphingosine and sphinganine were equivalent, consistent
with similar efficacies reported for these lipids with respect to
inhibition of PKC(14) . Conversely, the corresponding N-acyl derivatives ceramide and dihydroceramide differed
markedly in apoptotic capacity (Table 1), in that ceramide
potently induced DNA fragmentation, whereas dihydroceramide was
ineffective. Thus, while the effects of sphingosine have been
attributed to conversion to ceramide in some settings(23) , the
identical responses to sphingosine and sphinganine indicates that the
lethal actions of sphingoid bases do not reflect artifactual
accumulation of ceramide. This was confirmed in related studies
involving the mycotoxin fumonisin B
The apoptotic
capacity of sphingosine did not exhibit stereospecificity (Table 2), consistent with a specific involvement of PKC. Direct
comparison of D-erythro-sphingosine with L-erythro-sphingosine and the corresponding
enantiomer pair L-threo-sphingosine and D-threo-sphingosine revealed similar efficacies with
respect to induction of apoptotic DNA damage. Both the accumulation of
DNA fragments and breakage of bulk DNA in response to each isomer were
equivalent, although the L-threo isomer frequently
exhibited a slightly higher efficacy for this response (
Figure 4:
Concentration-response characteristics of
sphingosine action: quantitative studies. HL-60 cells were exposed to
sphingosine (So) over a broad range of concentrations
(0.001-100 µM) for 6 h. Multiple aspects of
apoptosis were then quantified as before. Panel A, clonogenic
capacity (
Figure 5:
Concentration-response characteristics of
sphingosine action: qualitative studies. HL-60 cells were exposed to
sphingosine (So) over a broad range of concentrations
(0.001-100 µM) for 6 h. Apoptotic DNA fragments were
then separated on agarose gels as before. Panel A, resolution
of DNA loop fragments by pulsed-field electrophoresis. Panel
B, resolution of oligonucleosomal DNA fragments by static-field
electrophoresis. Data shown are from a representative study performed
six times with comparable results.
Figure 6:
Potentiation of ceramide-induced DNA
damage by sphingoid bases. HL-60 cells were exposed to ceramide (Cer) in the absence or presence of sphingosine (So;
10 µM) or sphinganine (Sa; 10 µM)
for 6 h. Apoptotic DNA damage was then assessed by quantitative
spectrofluorophotometry as before. Panel A, formation (single-hatched bars) and release (double-hatched
bars) of double-stranded DNA fragments; values are expressed as
nanograms of DNA/10
Figure 7:
Potentiation of ceramide-induced apoptosis
by sphingosine. HL-60 cells were exposed to ceramide (0.0001 to 100
µM) in the absence (
Figure 8:
Potentiation of sphingomyelinase-induced
apoptosis by pharmacological inhibitors of PKC. HL-60 cells were
exposed to bacterial SMase (0.001-100 milliunits/ml) in the
absence (
Figure 9:
Potentiation of sphingomyelinase-induced
apoptosis by down-modulation of PKC. HL-60 cells were treated with
synthetic ceramide (N-octanoylsphingosine (C
The response to SMase
was markedly augmented by calphostin C, which acts at the
enzyme's regulatory domain (10 nM; Fig. 8A) or chelerythrine, which acts at the
enzyme's catalytic site (1 µM; Fig. 8B). Calphostin C and chelerythrine both produced
marked leftward shifts in the concentration-response profile to SMase.
The potentiative actions of these compounds differed in other respects,
however, inasmuch as the magnitude of the response to SMase at maximal
concentrations was significantly (p < 0.001) enhanced by
calphostin C, but not by chelerythrine. Thus, whereas both agents
increased SMase potency, only calphostin increased SMase efficacy. Furthermore, the induction of DNA fragmentation by synthetic
ceramide was sharply potentiated by chronic (i.e. 24 h)
pre-exposure to the non-tumor-promoting PKC activator bryostatin 1 (250
nM; Fig. 9), enhancing the response to ceramide by 89%.
These interactions were accompanied by extensive down-modulation of
total assayable PKC activity in crude cell lysates (Fig. 9, inset). PKC down-modulation was confirmed in parallel studies
in which expression of cPKC Sphingoid bases represent a versatile class of endogenous
inhibitory effectors of the PKC isoenzyme
family(13, 14) , and thus have been found to suppress
or attenuate numerous PKC-dependent aspects of leukemic cell survival.
In HL-60 cells, sphingosine and sphinganine markedly limit
proliferative capacity and viability(15) , and recent evidence
has suggested that this response involves the induction of
apoptosis(16) . Monocytoid differentiation in HL-60 cells is
sustained by PKC activity (reviewed in (25) ), a well defined
process elicited by prolonged treatment with synthetic
diglyceride(26) , bacterial phospholipase C(27) , or
tumor-promoting
phorboids(28, 29, 30, 31, 32) .
These responses are potently antagonized by sphingoid bases. Induction
of HL-60 cell differentiation by synthetic diglyceride is abolished by
sphinganine(33) . Phorboid-related maturation in these cells is
similarly attenuated by both sphinganine (33, 34) and
sphingosine(35) , as well as by such diverse pharmacological
inhibitors of PKC as isoquinoline derivatives (e.g. H7)(36) , fungal metabolites (e.g. staurosporine), and acylcarnitines (e.g. palmitoylcarnitine) (37) . Moreover, terminal monocytoid
differentiation of HL-60 cells ultimately culminates in apoptotic cell
death(38, 39) . This process reportedly results from
progressive, age-related increases in the intracellular availability of
sphingosine, the apparent consequence of an augmented capacity to
deacylate endogenous ceramide(16) . Whether such alterations in
sphingolipid metabolism represent an intrinsic feature of cellular
maturation, or instead reflect a generalized feedback response to the
sustained PKC activity necessary to support terminal differentiation,
remains to be determined. The present results demonstrate that
sphingoid bases exert both direct and indirect apoptotic influences in
myeloid leukemia cells. Acute exposure to sphingosine or sphinganine
were found to (a) induce double-stranded degradation of
genomic DNA, (b) suppress proliferative capacity, and (c) promote apoptotic cytoarchitectural changes. These
findings are in agreement with qualitative characterizations of
sphingosine-related apoptosis in HL-60 cells within the context of
terminal differentiation described by Ohta and co-workers(16) .
The apoptotic actions of sphingosine and sphinganine exhibited
essentially identical concentration-response profiles. A fundamental
change in DNA damage was noted at high sphingoid base concentrations (i.e. 10-25 µM), however. Specifically,
whereas bulk chromatin was continuously cleaved into large
( The intrinsic capacity of sphingoid
bases to initiate apoptosis is directly consistent with the central
cytoprotective role for the PKC isoenzyme family in the regulation of
leukemic cell survival proposed in previous
studies(1, 2, 12) . Nonetheless, these
findings must be interpreted with caution because the cellular
concentrations of these lipids required for maximal inhibition of PKC
activity may not be realized in living systems, an issue that has
received comment from other investigators(14, 42) .
The additional finding that sphingoid bases markedly potentiate the
induction of apoptosis by ceramide when present at sublethal levels may
therefore have considerable physiological significance. From a
mechanistic standpoint, this interaction is consistent with the
reciprocal ability of diglyceride to attenuate ceramide action that we
have described previously(2) . Taken together, these findings
raise the possibility that inhibition of PKC activity by endogenous
sphingoid bases contributes to the regulation of apoptosis, not by
initiating cell death directly, but rather by sensitizing the
intracellular signaling systems that govern cell survival to the
actions of a primary lethal messenger such as ceramide. Susceptibility
to the apoptotic influence of ceramide thus may represent a function of
the relative intracellular availability of sphingoid bases and
diradylglycerols. Studies designed to evaluate the impact of this
concentration ratio on the apoptotic efficacy of ceramide are currently
under way in our laboratory. Although the PKC isoenzyme family
represents a principal intracellular target for sphingoid
bases(13, 14) , there is ample evidence to indicate
that the bioeffector properties of these lipids may involve the
modulation of additional regulatory systems. For example, recent
investigation in other laboratories has demonstrated the existence of a
novel family of sphingosine-activated protein
kinases(43, 44) ; these isoenzymes reportedly are (a) stimulated by sphingosine in a highly stereospecific
manner (with a marked preference for the D-erythro species), but (b) completely insensitive to sphinganine.
Similarly, pronounced stereoselectivity is also associated with other
biological actions of sphingosine, including dephosphorylation of
pRb(45, 46) , and the inhibition of the c-Src/v-Src
protein kinases (47) and a variety of enzymatic activities that
require calmodulin for optimal function (e.g. the
multifunctional Ca While the biological actions of sphingosine have
been attributed, under some circumstances, to N-acylation of
sphingosine to form ceramide via the ceramide synthase
pathway(23) , the cytotoxic properties of sphingosine described
in this report are unlikely to stem from such a process. First, as
already noted, sphingosine and sphinganine exhibited equivalent potency
and efficacy in both the direct induction of apoptosis and the
potentiation of ceramide-dependent cell death. Conversion of
sphingosine and sphinganine (i.e. dihydrosphingosine) to the
corresponding N-acylated derivatives (i.e. ceramide
and dihydroceramide, respectively) yields metabolites with disparate
biological actions because the established bioeffector properties of
ceramide, including the capacity to induce apoptosis, reportedly are
not associated with dihydroceramide(3, 52) . Second,
and more significantly, both direct and indirect apoptotic influences
of sphingosine were unaffected by the mycotoxin fumonisin
B The capacity of sphingoid
bases to induce apoptosis is consistent with previous findings from
this and other laboratories demonstrating that diverse exogenous
inhibitors of PKC alone initiate this
process(12, 56, 57) . These results are also
compatible with other studies indicating that the apoptotic efficacy of
the potent antileukemic agent
1-[ In
conclusion, these observations demonstrate that sphingoid bases promote
apoptotic cell death in human myeloid leukemia cells through both
direct and indirect mechanisms. Within the context of physiological
regulation of apoptosis, the potentiation of ceramide-induced cell
death by sphingoid bases directly complements our previous observations
that diglyceride opposes ceramide action. On the basis of these
findings, therefore, it is proposed that (a) the regulation of
leukemic cell survival depends upon a balance between ceramide-driven
systems (e.g. ceramide-activated protein kinase) and PKC, and
that (b) the cytoprotective influence of PKC is modulated by
the reciprocal actions of sphingoid bases and diradylglycerols.
Volume 271,
Number 14,
Issue of April 5, 1996 pp. 8275-8284
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
750 nM), however,
sphingoid bases synergistically augmented the apoptotic capacity of
ceramide (10 µM), producing both a leftward shift in the
ceramide concentration-response profile and a pronounced increase in
the response to maximally effective levels of ceramide. Thus,
sphingosine and sphinganine increased both the potency and efficacy of
ceramide. The apoptotic capacity of bacterial sphingomyelinase (50
milliunits/ml) was similarly enhanced by either (a) acute
co-exposure to highly selective pharmacological inhibitors of protein
kinase C such as calphostin C and chelerythrine or (b) chronic
pre-exposure to the non-tumor-promoting protein kinase C activator
bryostatin 1, which completely down-modulated total assayable protein
kinase C activity. These findings demonstrate that inhibition of
protein kinase C by physiological or pharmacological agents potentiates
the lethal actions of ceramide in human leukemia cells, providing
further support for the emerging concept of a cytoprotective function
of the protein kinase C isoenzyme family in the regulation of leukemic
cell survival.
)is supported by several
lines of evidence. Increased intracellular availability of diglyceride
abrogates the initiation of apoptotic DNA damage by ceramide in both
HL-60 and U937 cells(1, 2) ; this effect is mimicked
by such diverse pharmacological PKC activators as the stage 1 tumor
promoters phorbol dibutyrate (2) and phorbol myristate acetate (2, 3) , the stage 2 tumor promoter
mezerein(2) , and the non-tumor-promoting macrocyclic lactone
bryostatin 1(2) . Collectively, these findings have defined
opposing cytotoxic and cytoprotective roles for ceramide and
diglyceride and, by extension, for their respective target enzymes in
the regulation of leukemic cell survival.
Drugs and Reagents
Synthetic preparations of D-erythro-sphingosine and D-erythro-sphinganine were obtained from Biomol
Research Laboratories, Inc. (Plymouth Meeting, PA). Other sphingosine
derivatives (e.g. 3-keto-D-erythro-sphingosine, N,N-dimethylsphingosine), and synthetic short-chain
preparations of ceramide (N-octanoylsphingosine) and
dihydroceramide (N-octanoylsphinganine) were also obtained
from Biomol. Synthetic diglyceride analogs
(1,2-dioctanoyl-sn-glycerol,
2,3-dioctanoyl-sn-glycerol, and
1,3-dioctanoyl-rac-glycerol) were obtained from Sigma. All
lipids were initially dissolved in 100% ethanol and stored at -70
°C. For experimental use, concentrated ethanol stocks of various
sphingolipids were complexed at a 1:1 molar ratio with delipidated
bovine serum albumin (fraction V; 2 mM in PBS) by vigorous
mixing for 90 min at 37 °C; stable protein-bound sphingolipid
preparations were stored at -20 °C. In contrast,
glycerolipids were used directly as concentrated stocks in 100%
ethanol. Bacterial preparations of sphingomyelinase (SMase; from Staphylococcus aureus) in a vehicle of 50% glycerol, 0.25 M Na
HPO
, pH 7.5, were obtained from
Sigma or from Biomol and stored at 4 °C. The selective PKC
inhibitors calphostin C and chelerythrine (LC Services Corporation,
Woburn, MA) were dissolved in sterile water, and stored at 4 °C.
The mycotoxin fumonisin B (Sigma) was dissolved in 100%
ethanol immediately before use. Bryostatin 1 was obtained in
lyophilized preparations from (a) Dr. George R. Pettit
(Arizona State University, Tempe, AZ) or from (b) the Cancer
Treatment Evaluation Program of the National Cancer Institute;
bryostatin 1 was dissolved in sterile MeSO and stored at
-20 °C. All test reagents were diluted to final
concentrations in complete medium at 37 °C; the vehicles used were
without discernible effect in HL-60 and U937 cells.Preparation of Sphingosine Stereoisomers
Various sphingosine stereoisomers were synthesized and
purified as described
previously(17, 18, 19) . D-erythro-sphingosine and L-erythro-sphingosine were prepared, respectively,
from tert-butoxycarbonyl-L-serine and tert-butoxycarbonyl-D-serine via coupling of the
Garner aldehyde (17) with lithium pentadecyne in
hexamethylphosphoramide-tetrahydrofuran as described by
Herold(18) , followed by Birch reduction in lithium-ethylamine
as described by Garner(19) . D- and L-threo-isomers were prepared in a similar manner,
with the exception that coupling reactions were performed with lithium
pentadecyne in the presence of zinc bromide in diethyl
ether(18) . Stereoisomers were characterized as N-biphenyl-carboxamido derivatives of sphingosine by high
performance liquid chromatography on the basis of elution from a chiral
column in hexane/2-propanol (8:2, v/v).Cell Culture
The human promyelocytic leukemic cell line HL-60 was derived
from a patient with acute promyelocytic
leukemia(20, 21) . The human monoblastic leukemia cell
line U937 was derived from a patient with diffuse histiocytic
lymphoma(22) . Both cell lines were grown in complete RPMI 1640
medium (phenol red-free formulation, supplemented with 1.0% sodium
pyruvate, non-essential amino acids, L-glutamine, penicillin,
and streptomycin (all from Life Technologies, Inc.) and 10%
heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT).
HL-60 and U937 cultures were passed twice weekly, both exhibiting
characteristic doubling times of 
24 h. Cultures were maintained
under a fully humidified atmosphere of 95% room air, 5% CO at 37 °C. Cell densities were determined by Coulter counter,
and basal cell viability was assessed by trypan blue exclusion.Test Exposures
All experimental incubations were performed as described
previously(1, 2, 12) . Cells in log-phase
growth were pelleted, washed twice in complete medium, resuspended at a
density of 4.25 
10
cells/ml), and maintained as
indicated above; in view of the well established sensitivity of
sphingoid base actions to surface dilution phenomena(13) , cell
density was carefully controlled in all test incubations. Cells were
exposed to test agents for appropriate intervals in complete medium;
loss of cells under these conditions due to either washing or cell
adherence was negligible (5%). Test incubations were terminated
with gentle pelleting of the cells by centrifugation at 400 g for 10 min at 4 °C; in most instances, aliquots of the
medium were retained for direct assay of released DNA fragments.
Following determination of cell density, the cells were pelleted and
prepared as outlined below for agarose gel electrophoresis,
spectrofluorophotometric assays of DNA fragments and DNA strand breaks,
assay of cloning efficiency, or examination of cellular morphology.Qualitative Analyses of DNA Damage
To assess both early and late aspects of ceramide-related DNA
degradation, apoptotic DNA fragments of greatly differing sizes were
resolved electrophoretically in parallel studies on both pulsed-field
and static-field agarose gels as follows.Pulsed-field Gel Electrophoresis
The formation of
rosette (300-kbp) and loop (50-kbp) DNA fragments was
assessed by field-inversion gel electrophoresis as described
previously(2) . Pelleted cells were resuspended in PBS and
mixed with molten 1.0% low melting-point agarose (In-Cert; FMC Corp.
Bioproducts), yielding a final concentration of 2 10
cells/ml; fractions of these mixtures (corresponding to 2
10
cells) were cast into precooled 85-µl block
molds and allowed to solidify at 4 °C. The agarose-imbedded lysates
were then treated with 250 mM EGTA, 250 mM EDTA, 1% N-lauroylsarcosine, pH 8.0 containing proteinase-K (200
µg/ml; Sigma) at 55 °C for 48 h. Deproteinated lysate plugs
were rinsed in 250 mM EDTA, 250 mM EGTA, pH 8.0, and
imbedded into 2.25% agarose gels (Sea-Kem Gold; FMC); high molecular
weight DNA fragments were resolved by field-inversion electrophoresis
at 6 V/cm for 24-28 h in 0.5 Tris borate/EGTA buffer at
14 °C; pulse intervals were ramped from t = 0.5 s to t = 50.0 s, with an F/R ratio of 3.0. Gels were stained for 6 h in 0.5
Tris borate/EGTA buffer containing ethidium bromide (0.5
µg/ml), and DNA fragments were visualized by UV transillumination.
DNA molecular weight reference preparations (48.6-kbp ladder; Life
Technologies, Inc.) routinely were run in parallel to facilitate
estimation of the size of rosette and loop DNA fragments.Static-field Gel Electrophoresis
The formation of
oligonucleosomal DNA fragments (0.2-1.2 kbp) was assessed by
conventional agarose gel electrophoresis as described
previously(2) . Pelleted cells were resuspended in PBS and
lysed by addition of 10 mM Tris-HCl, 15 mM EGTA, 15
mM EDTA, 0.1% Nonidet P-40, pH 7.4, yielding a final
concentration of 4 10 cells/ml; the lysates were
then treated with proteinase-K (200 µg/ml; Sigma) at 55 °C for
24 h. The deproteinated extracts were centrifuged at 45,000 g for 75 min at 4 °C, and the pellets were discarded; the
supernatants were subsequently treated with ribonuclease-A (100
µg/ml; Sigma) at 37 °C for 18 h. Aliquots of final lysate
preparations (corresponding to 2 10 cells) were
loaded into 2.25% agarose gels (Metaphor; FMC) impregnated with
ethidium bromide (0.5 µg/ml); low molecular weight DNA fragments
were resolved by electrophoresis at 6 V/cm for 90-180 min in 1
Tris acetate/EGTA buffer at 10 °C. DNA fragments were
visualized by UV transillumination. DNA molecular weight reference
preparations (100-bp ladder; Life Technologies, Inc.) were run in
parallel to facilitate estimation of the size of oligonucleosomal DNA
fragments.Quantitative Analyses of DNA Damage
The formation and release of DNA fragments, and the
corresponding breakage of bulk DNA were assessed as described
previously(1, 2) . To measure intracellular DNA
fragments, pelleted cells (4 10 cells/pellet in
quadruplicate) were resuspended in PBS and lysed by addition of 5
mM Tris-HCl, 30 mM EGTA, 30 mM EDTA, 0.1%
Triton X-100 (fully reduced), pH 8.0 (yielding a final density of
10 cells/ml), with gentle mechanical agitation. The lysates
were centrifuged at 45,000 g at 4 °C for 40 min;
to measure extracellular DNA fragments, aliquots of incubation medium
were adjusted to 5 mM Tris-HCl, 30 mM EGTA, 30 mM EDTA, pH 8.0, and centrifuged at 20,000 g at 4
°C for 40 min. The pellets were discarded, and the presence of
non-sedimenting DNA fragments in the supernatant from lysate and medium
extracts was determined by dilution in modified Tris-sodium/EGTA buffer
(3 mM NaCl, 10 mM Tris-HCl, 1 mM EGTA, pH
8.0) containing 1.0 µg/ml bis-benzimide trihydrochloride
(Hoechst 33258; Sigma), and monitoring net fluorescence in each sample
(
= 365, 
= 460).
Final DNA values were calculated relative to highly purified calf
thymus DNA calibration standard; values for all such responses are
uniformly expressed as nanograms/micrograms DNA recovered or released
from 10 cells, and reflect the absolute amount of
non-sedimenting, low molecular weight fragments of DNA present in
lysate and medium preparations. Corresponding loss of integrity of bulk
DNA was determined by enhanced-fluorescence alkaline unwinding analysis
as described previously(1, 2) . Pelleted cells (8.25
10 cells/pellet in quadruplicate) were resuspended
in cold PBS and subjected to timed alkaline denaturation in 0.1 N NaOH; denaturation was terminated by neutralization in 0.1 N HCl. Cells were then further diluted in PBS and lysed by addition
of 200 mM KHPO, 50 mM EDTA,
0.16% N-lauroylsarcosine with brief sonication. Damage to bulk
DNA in cell lysates was quantified by spectrofluorophotometry in the
presence of Hoechst 33258 ( = 350,
= 450); induction of strand breaks was
demonstrated by reduction of net DNA fluorescence. Values were
standardized against graded DNA strand-breakage induced by scaled
irradiation from a [
Cs] point source
(30-3000 rads), and are expressed as rad-equivalents.Clonogenic Assay
Because we have found that HL-60 cells resist uptake of
trypan blue even in advanced stages of apoptosis, the use of dye
exclusion was precluded in these studies as a valid index of diminished
viability; proliferative capacity was instead assessed in terms of
clonogenic potential. Pelleted cells were washed extensively and
prepared for soft-agar cloning as described
previously(1, 2, 12) . Cells were resuspended
in cold PBS and seeded in 35-mm culture plates at a fixed density (400
cell/ml/well) in complete RPMI 1640 medium containing 20% fetal calf
serum, 10% 5637-CM, and 0.3% Bacto agar. Cultures were maintained for
10-12 days, and formation of colonies (defined as groups of
50 cells) was scored using an inverted microscope.Cytology
Pelleted cells were resuspended in PBS, fixed in conventional
cytocentrifuge preparations, stained with 20% Wright-Giemsa stain, and
reviewed by light microscopy. The occurrence and mode of cell death in
each treatment group were determined based on morphological criteria
outlined previously(1, 2, 12) . At least 3
fields of 100 cells each were scored for each treatment by assessing
the expression of cytoarchitectural characteristics of either apoptosis (cell shrinkage, condensation of nucleoplasm and
cytoplasm, formation of membrane blebs and apoptotic bodies) or necrosis (cell swelling, nuclear expansion, deterioration of
organellar membranes, gross cytolysis).In Vitro Assay of Total Cellular Protein Kinase C
Activity
Assay of total protein kinase C activity in crude cell
homogenates was performed as described previously(58) .
Briefly, preparations of cell lysates were transferred to acetylated
filter discs and added to reactions mixtures containing lysis buffer
(20 mM Tris-HCl, 500 µM EDTA, 500 µM EGTA, pH 7.5), synthetic phospholipid, phorbol 12-myristate
13-acetate, and synthetic substrate (acetylated myelin basic protein N-terminal peptide AcMBP
). The reaction
was initiated by the addition of 25 µCi of
[
-
P]ATP, 20 µM non-isotopic
ATP, allowed to proceed for 5 min at 30 °C, and terminated by
addition of cold ortho-phosphoric acid (1%, v/v). The filters
were washed and radioactivity determined by conventional liquid
scintillometry.Western Analysis of cPKC
Cells were lysed in 2 
Expression Laemmli buffer, sonicated
briefly, and stored at -20 °C pending analysis. Cellular
proteins (2 10 cell equivalents/condition) were
resolved by electrophoresis on 12.5% polyacrylamide gels, and then
transferred to nitrocellulose membranes. Membranes were sequentially
incubated in (a) rabbit anti-human polyclonal antibody
(1:5000; Santa Cruz) for 1 h and (b) goat anti-rabbit
polyclonal antibody horseradish peroxidase conjugate (1:5000;
Calbiochem) for 1 h; immunoreactive cPKC was visualized by
enhanced chemiluminescence.
Induction of Apoptosis by Sphingosine and
Sphinganine
Apoptotic cell death in HL-60 cells is characterized
by loss of proliferative capacity, double-stranded degradation of
genomic DNA, and profound alterations of cellular morphology. Highly
selective pharmacological PKC inhibitors induce apoptosis in HL-60
cells(12) , raising the possibility that sphingoid bases
mediate similar lethal influences in the physiological regulation of
cell death. The capacity of sphingoid bases to promote apoptotic cell
death therefore was examined in these cells. Exposure of HL-60 cells to
synthetic preparations of sphingosine or sphinganine at a fixed
concentration (10 µM) for 6 h potently induced apoptosis.
Qualitative assessment of DNA damage on agarose gels demonstrated
electrophoretic patterns of DNA fragments formed by internucleosomal
hydrolysis of static chromatin in response to either lipid (Fig. 1); these included both high molecular weight loop
fragments (appearing as single truncated bands of 50 kbp on
pulsed-field gels; Fig. 1A) and low molecular weight
oligonucleosomal fragments (appearing as ``ladders'' of
0.2-1.2 kbp on static-field gels; Fig. 1B).
In related studies, quantitative assessment of DNA damage by
spectrofluorophotometry demonstrated extensive degradation of genomic
DNA in response to sphingosine and sphinganine (Fig. 2). Both
lipids comparably promoted the formation and release of double-stranded
DNA fragments (Fig. 2A). Under basal conditions, such
fragments were present in intracellular levels of 225 ng/10 cells and in extracellular levels of 55 ng/10 cells. Sphingosine (10 µM) and sphinganine (10
µM) increased the total accumulation of apoptotic DNA
fragments respectively to 2206 ± 280 ng/10 cells (p < 0.001) and 2070 ± 158 ng/10 cells (p < 0.001). In addition, both lipids promoted extensive
breakage of bulk DNA (Fig. 2B). Spontaneous breakage of
bulk DNA was detected at a level of 155 rad equivalents.
Sphingosine (10 µM) and sphinganine (10 µM)
increased the extent of bulk DNA breakage, respectively, to 5387
± 195 rad equivalents and to 5009 ± 145 rad equivalents.
Sphingosine (10 µM) and sphinganine (10 µM)
also substantially suppressed the proliferative capacity of HL-60
cells, such that colony formation was reduced by 87% and 82%,
respectively (data not shown). In addition, while apoptotic morphology
was discernible in 1.5% of vehicle-treated cells, both lipids
elicited the expression of morphological features classically
associated with apoptosis, including condensed nucleoplasm and
cytoplasm, formation of membrane blebs and apoptotic bodies,
fragmentation of the nucleus, and overall cell shrinkage. Exposure to
sphingosine (10 µM) or sphinganine (10 µM)
for 6 h increased the fraction of cells exhibiting apoptotic traits to
94% and 91%, respectively (Fig. 3). Comparable apoptotic
responses to sphingosine and sphinganine were also observed in parallel
studies with U937 cells (data not shown).
50 kbp) DNA fragments by pulsed-field
electrophoresis. Panel B, resolution of oligonucleosomal DNA
fragments (0.2-1.2 kbp) by static-field electrophoresis.
Data shown are from a representative study performed four times with
comparable results.
cells. Panel
B, loss of integrity of bulk DNA (solid bars); values are
expressed as rad equivalents. Data shown are from a representative
study performed four times with comparable results. All values reflect
mean ± S.E. of quadruplicate
determinations.
, which prevents N-acylation of sphingoid bases by inhibition of ceramide
synthase(24) . There was no evidence of apoptotic DNA damage
following exposure of HL-60 cells to fumonisin B (100
µM) for 6 h; moreover, the extent of DNA fragmentation
elicited by exposure to sphingosine (10 µM) or sphinganine
(10 µM) for 6 h was not attenuated in the presence of
fumonisin B (Table 1), confirming that sphingoid
base-related cell death was not mediated by ceramide.
15%).
Structurally related sphingoid bases were also screened for potential
apoptotic capacity in HL-60 cells (data not shown). For example, the
methylated derivative N,N-dimethylsphingosine was somewhat
more potent than sphingosine in the induction of apoptotic DNA damage (e.g. by 28%), whereas 3-ketosphingosine was essentially
ineffective at promoting apoptosis.
Concentration-response Characteristics of
Sphingosine-induced Apoptosis
The concentration-response
characteristics of sphingosine action were determined in subsequent
studies (Fig. 4). Exposure of HL-60 cells to sphingosine over a
broad range of concentrations (0.001-100 µM) for 6 h
deceased clonogenicity and increased expression of apoptotic
cytoarchitecture in a concentration-dependent manner (Fig. 4A). These responses were inversely correlated (R = 0.988). Both the loss of clonogenicity
and the appearance of apoptotic morphology were evident at 1 µM and maximal at 10 µM, with respective EC
values of 2.1 and 2.4 µM. The induction of apoptotic
DNA degradation, as reflected by the formation and release of DNA
fragments (Fig. 4B) and the corresponding breakage of
bulk DNA (Fig. 4C), exhibited divergent
concentration-response profiles. The concentration-response profile for
sphingosine-induced DNA fragmentation was distinctly biphasic,
reminiscent of the apoptotic responses to selective pharmacological PKC
inhibitors reported previously(12) . Significant (p < 0.01) accumulation of DNA fragments was discernible at 1
µM and maximal at 10 µM; above 10
µM, the generation of DNA fragments declined progressively
(to 40% of the maximal fragmentation observed at 10
µM). As the total number of cells recovered at the end of
the exposure interval was not appreciably diminished, the apparent
reduction in the extent of DNA damage associated with exposure to
sphingosine at higher levels (i.e. >10 µM)
could not be attributed to release of DNA upon physical dissolution of
dead cells. In contrast, the concentration-response profile for
sphingosine-induced breakage of bulk DNA was linear, rather than
biphasic. Significant (p<0.01) breakage of bulk DNA was detected at
0.1 µM and maximal at 25 µM; above 25
µM, however, DNA breakage appeared to remain constant. The
disparate concentration-response profiles for sphingosine provided by
these separate assays suggested a fundamental change in the nature of
DNA damage at higher sphingosine levels, a supposition that was
confirmed in electrophoretic analyses of apoptotic DNA fragments (Fig. 5). Concentration-related changes in the appearance of
apoptotic DNA fragments were demonstrated on both pulsed-field gels (Fig. 5A) and static-field gels (Fig. 5B). DNA loop fragments were initially observed
at 1 µM and persisted throughout the range of
concentrations tested; these bands increased in intensity and assumed a
more compact appearance as the sphingosine concentration was escalated
to 10 and 100 µM. In contrast, oligonucleosomal DNA
fragment ladders were observed exclusively at 10 µM, and
were replaced by a very faint continuous streak of DNA at 100
µM. Virtually identical electrophoretic profiles of
apoptotic DNA fragments were obtained from HL-60 cells in response to
sphinganine (not shown).
) and occurrence of apoptotic morphology (
),
expressed as % control colony formation and % total cells. Panel
B, spectrofluorophotometric determination of the formation
(
) and release (
) of DNA fragments, with calculated total
accumulation of DNA fragments (
); values are expressed as
micrograms of DNA/10 cells. Panel C,
spectrofluorophotometric determination of bulk DNA breakage ();
values are expressed as kilorad equivalents. All values reflect the
mean ± S.E. of quadruplicate determinations. Data shown are from
representative studies repeated four times with comparable
results.
Potentiation of Ceramide-induced Apoptosis by Sphingoid
Bases and Pharmacological Inhibitors of PKC
Previous
investigations have demonstrated that ceramide mediates the induction
of apoptotic cell death in HL-60 and U937
cells(1, 2, 3) . In addition, we have shown
that the apoptotic response to ceramide in these cells is attenuated or
abolished by diglyceride and a variety of pharmacological PKC
activators(1, 2) . To test the converse possibility
that ceramide-related apoptosis is potentiated by sphingoid bases,
additional studies were conducted to assess the apoptotic capacity of
ceramide in the absence or presence of sphingosine and sphinganine (Fig. 6). Exposure of HL-60 cells to ceramide at a maximally
effective concentration (10 µM) for 6 h potently induced
apoptotic DNA damage, increasing the net (i.e. intracellular
and extracellular) accumulation of double-stranded DNA fragments to
1635 ± 245 ng/10 cells and bulk DNA breakage to 3315
± 325 rad equivalents. Ceramide exposure also increased the
fraction of cells expressing apoptotic traits to 36% (data not shown).
Co-exposure to ceramide (10 µM) and either sphingosine or
sphinganine at a sublethal concentration (750 nM)
significantly (p < 0.001) enhanced ceramide action, as
reflected by both the net accumulation of DNA fragments and the
breakage of bulk DNA. In fact, the extent of ceramide-induced DNA
damage was augmented by approximately 89-96% in the presence of
either sphingoid base. Moreover, sphingosine and sphinganine increased
the fraction of cells exhibiting apoptotic morphology in response to
ceramide to 64% and 61%, respectively (data not shown).
Ceramide-induced apoptosis was comparably enhanced by sphingosine and
sphinganine in U937 cells (data not shown). In other studies, HL-60
cells were exposed to ceramide over a broad range of concentrations
(0.0001-100 µM) in the absence or presence of
sphingosine at a fixed (subeffective) concentration (750 nM)
for 9 h (Fig. 7). As reported previously (1) , ceramide
produced a linear concentration-related increase in the accumulation of
double-stranded DNA fragments. Sphingosine produced a distinct increase
in the response to ceramide, consisting of both (a) a marked
leftward shift in the ceramide concentration response profile and (b) a substantial increase in the effectiveness of maximal
ceramide levels; this interaction thus appeared to entail increases in
both the potency and efficacy of ceramide action. In other trials,
stereochemical aspects of the reciprocal modulation of
ceramide-mediated DNA degradation by sphingosine and diglyceride were
examined. HL-60 cells were exposed to ceramide at a maximal
concentration (10 µM) for 6 h in the absence or presence
of various isomers of sphingosine (750 nM) or diacylglycerol
(10 µM). Consistent with the direct induction of apoptosis
by sphingosine described above, potentiation of ceramide-related
apoptosis by sphingosine was not stereoselective (Table 3). When
compared directly, both the D- and L-forms of erythro and threo enantiomers of sphingosine
equivalently augmented the extent of DNA damage obtained following 6-h
exposure to ceramide. As we have noted previously, however(2) ,
the ability of diglyceride to limit ceramide-induced DNA damage was
exclusively associated with the 1,2-sn-substituted isomer,
whereas the 2,3-sn-substituted and
1,3-rac-substituted species were ineffective (Table 4).
These steric aspects of the reciprocal modulation of ceramide action by
sphingosine and diglyceride are consistent with alterations in PKC
activity.
cells. Panel B, loss of
integrity of bulk DNA (solid bars); values are expressed as
rad equivalents. Data shown are from a representative study performed
four times with comparable results. All values reflect mean ±
S.E. of quadruplicate determinations.
) or presence () of
sphingosine (750 nM) for 6 h. The total accumulation of
apoptotic DNA fragments was then assessed by quantitative
spectrofluorophotometry as before; values are expressed as micrograms
of DNA/10 cells. Data shown are from a representative study
performed four times with comparable results. All values reflect mean
± S.E. of triplicate determinations.
Effects of Pharmacological Protein Kinase C Modulators on
Ceramide-induced DNA Fragmentation
In other studies,
potentiation of ceramide-induced apoptosis was also observed following
treatment with bacterial sphingomyelinase (SMase; 0.001-100
milliunits/ml) by either (a) acute co-exposure to highly
selective pharmacological PKC inhibitors such as calphostin C or
chelerythrine at concentrations previously shown to be sub-effective in
the induction of DNA fragmentation(12) , or (b)
following chronic pre-exposure to a non-differentiation-inducing
pharmacological PKC activator such as bryostatin 1 at a concentration
sufficient to promote extensive down-modulation of assayable PKC
activity (58) ( Fig. 8and Fig. 9). As we have
described in previous reports(1, 2) , treatment of
HL-60 cells with SMase induced the accumulation of double-stranded DNA
fragments in a concentration-dependent manner.
) or presence () of either calphostin C (panel A) or chelerythrine (panel B) for 6 h. The
total accumulation of apoptotic DNA fragments was then assessed by
quantitative spectrofluorophotometry as before; values are expressed as
micrograms of DNA/10 cells. Data shown are from a
representative study performed four times with comparable results. All
values reflect mean ± S.E. of triplicate
determinations.
Cer); 10 µM) for
9 h following pretreatment with either vehicle (Veh) or
bryostatin 1 (BRY, 250 nM) for 24 h. Total
accumulation of apoptotic DNA fragments was then assessed by
quantitative spectrofluorophotometry as before; values are expressed as
micrograms of DNA/10 cells. Data shown are from a
representative study performed three times with comparable results.
Values reflect mean ± S.E. of triplicate determinations. Inset, HL-60 cells were pretreated with vehicle (VEH)
or bryostatin 1 (BRY, 250 nM) for 24 h; total
cellular PKC activity was then determined by in vitro as
described under ``Experimental Procedures.'' Data shown are
from a representative study performed three times with comparable
results. Values reflect mean ± S.E. of triplicate
determinations.
, the predominant species of the enzyme
present in HL-60 cells, was monitored by conventional Western analysis
(data not shown); 24-h pre-exposure to bryostatin 1 virtually
eliminated the presence of immunoreactive cPKC. Neither
sphingosine nor sphinganine produced an additional augmentation of
ceramide-related DNA damage in HL-60 cells down-modulated for PKC
activity by bryostatin 1 pretreatment, however (data not shown),
consistent with the position that PKC represents the primary
subcellular target for sphingoid bases in the potentiation of ceramide
action.
50-kbp) DNA fragments, subsequent degradation of this high
molecular weight material into small (0.2-2.0-kbp)
oligonucleosomal fragments was arrested. This phenomenon presumably
reflects selective, concentration-related inhibition of the subtype(s)
of apoptotic endonuclease responsible for internucleosomal hydrolysis
of 50-kbp fragments. While such an underlying mechanism has yet to be
demonstrated conclusively, this observation is consistent with reports
suggesting that very early genomic lesions such as the initial breakage
of static chromatin into high molecular weight (i.e. 300- and
50-kbp) DNA fragments are more central to the apoptotic process than
the subsequent formation of low molecular weight DNA cleavage products (i.e. 0.2-2.0-kbp oligonucleosomal
ladders)(40, 41) . Moreover, it is noteworthy that a
similar concentration-dependent change in the character of apoptotic
DNA damage has been previously documented in HL-60 cells following
exposure to highly selective PKC inhibitors such as calphostin C and
chelerythrine(12) .
-/calmodulin-dependent protein
kinase) (48) . Nonetheless, the stereoselectivity of sphingoid
base action described in these studies is most consistent the
established lipid sensitivity of PKC, strongly suggesting that the
apoptotic properties of sphingosine and sphinganine derive from
inhibition of PKC. First, whereas only the D-erythro species occurs naturally in mammalian systems(49) , the
four isomers are equipotent in the inhibition PKC activity in
vitro(35) , and we observed a complete lack of
stereoselectivity in the capacity of sphingosine to initiate apoptosis.
Second, sphingosine and sphinganine are equivalent inhibitors of PKC
(suggesting that the trans-4 double bond is not essential for
inhibition of PKC activity by sphingoid bases) (35) , and we
noted essentially identical apoptotic responses to sphingosine and
sphinganine. An analogous relationship has been noted with respect to
the physiological activation of PKC by diglycerides, in that
1,2-diradyl-sn-glycerols are stimulatory, whereas
1,3-rac-substituted species are
inactive(50, 51) . The application of such steric
influences as criteria for implicating PKC in the mechanism of action
of sphingoid bases and diradylglycerols also appears to be relevant in
considering the modulation of ceramide action. Thus, the findings that
the apoptotic capacity of ceramide was (a) comparably
augmented by both D- and L- forms of erythro-sphingosine and threo-sphingosine, but (b) selectively abolished by sn-1,2-substituted (but
not sn-2,3-substituted or rac-1,3-substituted) forms
of diglyceride additionally supports an involvement of PKC activity in
the reciprocal modulation of ceramide action by sphingosine and
diglyceride. Also consistent with an involvement of PKC in the
apoptotic properties of sphingoid bases, down-modulation of PKC by
chronic pre-exposure to bryostatin 1 potentiated ceramide-induced
apoptosis to essentially the same extent as did acute inhibition of PKC
by sphingoid bases. In this regard, it is significant that the
potentiated response to ceramide noted in PKC-down-modulated cells
could not be further augmented in the presence of sphingosine
or sphinganine.. Because this toxin prevents the acylation of sphingosine
to ceramide though competitive inhibition of ceramide synthase ( (53) and (54) ; reviewed in (24) ), the actions
of sphingosine described above more likely to reflect a direct action
of sphingosine, rather than the artifactual accumulation of ceramide.
Finally, it should be noted that, whereas a recent report describes
transcriptional repression of multiple PKC isoforms in CV-1 monkey
kidney cells following chronic treatment with fumonisin
B(55) , we found no evidence that acute (i.e. 6-h) exposure to 100 µM fumonisin B induced apoptosis in HL-60 cells.
-D-arabinofuranosyl]cytosine is augmented by
manipulations that reduce cellular PKC activity, including both (a) down-modulation of PKC by chronic exposure to
pharmacological PKC activators (58) and (b) inhibition
of PKC by acute exposure to pharmacological PKC
inhibitors(59) . Furthermore, preliminary observations indicate
that the ability of
1-[-D-arabinofuranosyl]cytosine to induce
apoptosis in HL-60 cells is also subject to reciprocal modulation by
diglyceride and sphingosine. ()Collectively, these findings
have potentially important implications for targeting PKC in the
development of novel antileukemic strategies. Indeed, the potential
utility of sphingoid bases as antineoplastic agents has been noted by
other investigators(60) . Antitumor actions of sphingosine and
structurally related compounds have been documented in numerous cell
types (reviewed in (61) ). For example, sphingosine and other
sphingoid bases profoundly reduce tumor cell number in vitro(62) and restrict tumor growth and metastasis in
vivo(63) . Similarly, synthetic structural analogs of
sphingoid bases (e.g. stearylamine) have been found to inhibit
the activity of PKC in purified preparations(33) , and to exert
potent antitumor influences both in vitro(64) and in vivo(65) . Furthermore, recent observations by
Schwartz and co-workers indicate that safingol (referred to elsewhere
as SPC-100270), a synthetic preparation of L-threo-sphinganine, potently limits the extent of
tumor cell invasiveness (66) and substantially augments the
antineoplastic actions of such diverse agents as doxorubicin and
mitomycin(67) . Whether these interactions stem from
potentiation of tumor cell apoptosis remains to be established.
))
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H. Nakamura, T. Oda, K. Hamada, T. Hirano, N. Shimizu, and H. Utiyama Survival by Mac-1-mediated Adherence and Anoikis in Phorbol Ester-treated HL-60 Cells J. Biol. Chem., June 19, 1998; 273(25): 15345 - 15351. [Abstract] [Full Text] [PDF]
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R. R. Dugyala, R. P. Sharma, M. Tsunoda, and R. T. Riley Tumor Necrosis Factor-alpha as a Contributor in Fumonisin B1 Toxicity J. Pharmacol. Exp. Ther., April 1, 1998; 285(1): 317 - 324. [Abstract] [Full Text]
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 A. Foghi, A. Ravandi, K. J. Teerds, H. van der Donk, A. Kuksis, and J. Dorrington Fas-Induced Apoptosis in Rat Thecal/Interstitial Cells Signals Through Sphingomyelin-Ceramide Pathway Endocrinology, April 1, 1998; 139(4): 2041 - 2047. [Abstract] [Full Text] [PDF]
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 K. L. Auer, J. Contessa, S. Brenz-Verca, L. Pirola, S. Rusconi, G. Cooper, A. Abo, M. P. Wymann, R. J. Davis, M. Birrer, et al. The Ras/Rac1/Cdc42/SEK/JNK/c-Jun Cascade Is a Key Pathway by Which Agonists Stimulate DNA Synthesis in Primary Cultures of Rat Hepatocytes Mol. Biol. Cell, March 1, 1998; 9(3): 561 - 573. [Abstract] [Full Text]
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G. P. Amarante-Mendes, C. Naekyung Kim, L. Liu, Y. Huang, C. L. Perkins, D. R. Green, and K. Bhalla Bcr-Abl Exerts Its Antiapoptotic Effect Against Diverse Apoptotic Stimuli Through Blockage of Mitochondrial Release of Cytochrome C and Activation of Caspase-3 Blood, March 1, 1998; 91(5): 1700 - 1705. [Abstract] [Full Text] [PDF]
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S. M. Vaingankar and M. Martins-Green Thrombin Aivation of the 9E3/CEF4 Chemokine Involves Tyrosine Kinases Including c-src and the Epidermal Growth Factor Receptor J. Biol. Chem., February 27, 1998; 273(9): 5226 - 5234. [Abstract] [Full Text] [PDF]
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 T. Ariga, W. D. Jarvis, and R. K. Yu Role of sphingolipid-mediated cell death in neurodegenerative diseases J. Lipid Res., January 1, 1998; 39(1): 1 - 16. [Abstract] [Full Text]
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R. Emkey and C. R. Kahn Cross-talk between Phorbol Ester-mediated Signaling and Tyrosine Kinase Proto-oncogenes. II COMPARISON OF PHORBOL ESTER AND SPHINGOMYELINASE-INDUCED PHOSPHORYLATION OF ErbB2 AND ErbB3 J. Biol. Chem., December 5, 1997; 272(49): 31182 - 31189. [Abstract] [Full Text] [PDF]
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W. D. Jarvis, F. A. Fornari Jr., K. L. Auer, A. J. Freemerman, E. Szabo, M. J. Birrer, C. R. Johnson, S. E. Barbour, P. Dent, and S. Grant Coordinate Regulation of Stress- and Mitogen-Activated Protein Kinases in the Apoptotic Actions of Ceramide and Sphingosine Mol. Pharmacol., December 1, 1997; 52(6): 935 - 947. [Abstract] [Full Text]
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D. A. Wiesner, J. P. Kilkus, A. R. Gottschalk, J. Quintans, and G. Dawson Anti-immunoglobulin-induced Apoptosis in WEHI 231Cells Involves the Slow Formation of Ceramide from Sphingomyelin and Is Blocked by bcl-xL J. Biol. Chem., April 11, 1997; 272(15): 9868 - 9876. [Abstract] [Full Text] [PDF]
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O. Cuvillier, L. Edsall, and S. Spiegel Involvement of Sphingosine in Mitochondria-dependent Fas-induced Apoptosis of Type II Jurkat T Cells J. Biol. Chem., May 19, 2000; 275(21): 15691 - 15700. [Abstract] [Full Text] [PDF]
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H. Wu, G. N. Rao, B. Dai, and P. Singh Autocrine Gastrins in Colon Cancer Cells Up-regulate Cytochrome c Oxidase Vb and Down-regulate Efflux of Cytochrome c and Activation of Caspase-3 J. Biol. Chem., October 13, 2000; 275(42): 32491 - 32498. [Abstract] [Full Text] [PDF]
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A. Zitzer, R. Bittman, C. A. Verbicky, R. K. Erukulla, S. Bhakdi, S. Weis, A. Valeva, and M. Palmer Coupling of Cholesterol and Cone-shaped Lipids in Bilayers Augments Membrane Permeabilization by the Cholesterol-specific Toxins Streptolysin O and Vibrio cholerae Cytolysin J. Biol. Chem., April 27, 2001; 276(18): 14628 - 14633. [Abstract] [Full Text] [PDF]
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