(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 (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.
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) ()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.
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 (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.
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 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.
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, 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.
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 (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.
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 () 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.
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 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.
Figure 7:
Potentiation of ceramide-induced apoptosis
by sphingosine. HL-60 cells were exposed to ceramide (0.0001 to 100
µM) in the absence () 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.
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 () 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.
Figure 9:
Potentiation of sphingomyelinase-induced
apoptosis by down-modulation of PKC. HL-60 cells were treated with
synthetic ceramide (N-octanoylsphingosine (CCer); 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 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, 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.
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
(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) .
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-/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.
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. 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.
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-[-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.
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.