(Received for publication, October 30, 1995; and in revised form, November 27, 1995)
From the
In our previous study, the sphingosine-like immunosuppressant, ISP-1, was found to suppress the proliferation of an interleukin-2-dependent cytotoxic T cell line, CTLL-2, through the inhibition of serine palmitoyltransferase, which catalyzes the committed step of sphingolipid biosynthesis. Analysis of the effect of ISP-1 by flow cytometry revealed that the ISP-1-dependent decrease in cell number was not due to inhibition of the cell cycle progression of CTLL-2 cells but to the induction of apoptosis of the cells. The ISP-1-induced apoptosis was inhibited by the addition of sphingosine (2 µM), suggesting that this ISP-1-induced apoptosis is triggered by the decrease in the intracellular levels of sphingolipids caused by the inhibition of serine palmitoyltransferase. However, another interleukin-2-dependent cell line, F7, which was derived from a mouse pro-B cell line, did not show ISP-1-dependent apoptosis, indicating that the effect of ISP-1 may be specific for a certain type of T cell lineage such as CTLL-2. On the other hand, a high dose of sphingosine (5 µM) by itself induced the apoptosis of CTLL-2 cells. This sphingosine-dependent apoptosis was also observed with F7 cells. These results provide evidence that the intracellular levels of sphingolipids play an important role in the signaling of the escape from and onset of apoptosis of CTLL-2 cells.
Several lines of evidence have suggested that sphingosines are
involved in apoptosis. Exogenously added cell-permeable ceramides
induce the apoptosis of U937 human leukemia cells (1) and
several other cell lines(2) . Later, a natural ceramide was
shown to induce apoptosis when added as a cell-permeable form in a
mixture of dodecane and ethanol(3) . Furthermore, the level of
intracellular ceramide was shown to increase through the degradation of
sphingomyelin under such conditions that apoptosis was induced by tumor
necrosis factor- (4, 5, 6) ,
-irradiation,(7) , and Fas
ligation(8, 9, 10) . Ceramide is, therefore,
proposed to be a second messenger for apoptosis. Recently, exogenously
added sphingosine was also demonstrated to induce the apoptosis of
human neutrophils (11) and HL-60 human leukemia cells (12) , and the intracellular level of sphingosine increased in
the cases of tumor necrosis factor-
-induced apoptosis of human
neutrophils (11) and phorbol ester-induced apoptosis of HL-60
cells(12) .
A new potent immunosuppressant, ISP-1, is a
fungal product that has a structure very similar to that of
sphingosine(13) . ISP-1 inhibited the mouse allogenic mixed
lymphocyte reaction and allo-reactive cytotoxic T lymphocyte generation in vivo, exhibiting a potency of 10-100-fold greater
than that of cyclosporin A, the most widely used immunosuppressant.
Unlike cyclosporin A and FK506 (another popular immunosuppressant),
ISP-1 did not interfere with IL-2 ()production in the mixed
lymphocyte reaction(13) , but instead it suppressed the
IL-2-dependent growth of the mouse cytotoxic T cell line,
CTLL-2(14) . This suppression is most probably triggered by a
reduction of the intracellular levels of sphingolipids due to the
inhibition of serine palmitoyltransferase, which catalyzes the first
step of sphingolipid biosynthesis, i.e. the condensation of
serine and palmitoyl-CoA into ketodihydrosphingosine(14) .
In this paper, we report that the overall growth suppression of CTLL-2 cells by ISP-1 is not due to inhibition of cell proliferation but to the apoptosis of the cells caused by inhibition of the de novo synthesis of sphingosine. This is the first evidence that apoptosis is caused by a reduction in the intracellular levels of sphingolipids.
Although ISP-1 was demonstrated to be a potent inhibitor of serine palmitoyltransferase, which catalyzes the first step of sphingolipid biosynthesis, i.e. the condensation of serine and palmitoyl-CoA into ketodihydrosphingosine(14) , the mechanism underlying the inhibition of cell growth by ISP-1 was not clear. In an attempt to elucidate the mechanism underlying the suppression of CTLL-2 cell growth by ISP-1, the effect of ISP-1 on cell cycle progression was investigated by the flow cytometric method. As shown in Fig. 1B, incubation of the cells in the presence of 47 nM ISP-1 for 48 h did not affect the cell cycle progression of CTLL-2 cells significantly but instead dramatically increased the hypodiploid DNA peaks, which are commonly found in apoptosis(19) . In order to confirm that the cells were indeed in the process of apoptosis, the chromosomal DNA profiles were investigated. As shown in Fig. 2A, lane 2, genomic DNA obtained from ISP-1-treated cells gave a ladder on agarose gel electrophoresis, which is a characteristic of apoptosis-specific internucleosomal DNA fragmentation(20) , while this fragmentation was not seen in mock-treated cells (lane 1). Further confirmation of apoptosis was the morphological changes of nuclei detected in ISP-1-treated cells. As shown in Fig. 3B, ISP-1-treated CTLL-2 cells showed morphological changes typical of apoptosis including condensed chromatin and fragmented nuclei revealed upon DNA staining with Hoechst 33342. All these results indicated that the ISP-1-dependent decrease in the number of CTLL-2 cells was not due to inhibition of the proliferation of CTLL-2 cells but to the acceleration of cell death by apoptosis.
Figure 1:
Flow cytometry analysis of propidium
iodide-stained CTLL-2 cells. Cells were cultured with the methanol
vehicle (A), 47 nM ISP-1 (B), 47 nM ISP-1 + 2 µM sphingosine (C), or 5
µM sphingosine (D) for 48 h (A, B, and C) or 8 h (D). After treatment, the cells were
stained and analyzed as described under ``Experimental
Procedures.'' The log scale DNA fluorescence intensity was
plotted. Bar 1, apoptotic cells; bar 2,
G/G
phase cells; bar 3, S phase cells; bar 4, G
/M phase cells. Insets, cell
cycle profiles were calculated from the number of nuclei in each cell
cycle phase using the linear scale plot of DNA fluorescence intensity
and expressed as a percentage of the total cells. The apoptotic cells
were excluded from the calculation.
Figure 2: Agarose gel electrophoresis of the fragmented DNA from CTLL-2 cells. A, CTLL-2 cells were cultured for 48 h with the methanol vehicle (lane 1) or 47 nM ISP-1 (lane 2). After treatment, the fragmented DNA was extracted under conditions with which intact genomic DNA is not extracted and analyzed as described under ``Experimental Procedures.'' B, CTLL-2 cells were treated with 47 nM ISP-1 for the indicated times. M, molecular weight markers (100-base pair ladder of DNA).
Figure 3: Morphological appearance of CTLL-2 cells treated with the methanol vehicle (A), 47 nM ISP-1 (B), 47 nM ISP-1 + 2 µM sphingosine (C), or 5 µM sphingosine (D) for 48 h (A, B, and C) or 8 h (D). After treatment, the cell nuclei were stained with Hoechst 33342 as described under ``Experimental Procedures.'' Bars indicate 10 µm.
In order to determine whether or not inhibition of the condensation of serine and palmitoyl-CoA into ketodihydrosphingosine is the direct trigger of the apoptosis of CTLL-2 cells, the effect of sphingosine, a compound located downstream of ketodihydrosphingosine in the sphingolipid biosynthesis pathway, on ISP-1-dependent apoptosis was studied. As shown in Fig. 1C, upon the addition of sphingosine (2 µM) to the incubation medium containing 47 nM ISP-1, the hypodiploid DNA peaks disappeared almost completely, which is in good agreement with the almost 100% recovery of cell proliferation with the same treatment previously reported(14) , suggesting again that the inhibition of cell growth by ISP-1 is caused by the progression of apoptosis due to the reagent.
The ability of sphingosine to counteract the effect of ISP-1 is stereoselective and cannot be mimicked by dimethylsphingosine(14) ; therefore, it is unlikely to be due to inhibition of protein kinase C, which does not show this structural selectivity(21, 22, 23) . These results suggested that exogenously added sphingosines did not function as inhibitors of protein kinase C and that ISP-1-induced apoptosis of CTLL-2 cells was triggered by the decrease in the intracellular levels of sphingolipids due to inhibition of serine palmitoyltransferase. The chemical nature of the downstream effector that is directly involved in apoptosis has not yet been identified. However, Fumonisin B1, which inhibits the conversion of sphingosine to ceramide(24) , did not inhibit the effect of exogenously added sphingosine on the ISP-1 action (14) , suggesting that ceramide is not the downstream effector and that sphingosine derivatives with a free amino group such as sphingosine and sphingosine 1-phosphate are possible candidates.
Incidentally, it was reported that exogenously added sphingosine
itself induced apoptosis in human neutrophils (11) and HL-60
promyelocytic leukemia cells (12) when it was administered at a
high concentration of 5-10 µM. This was also true
for CTLL-2 cells, as shown in Fig. 1D. Taken together,
these results suggest that both hypo- and hyperintracellular levels of
sphingolipids induced apoptosis of CTLL-2 cells and that the
maintenance of the proper levels of these compounds is critically
important for the cells to escape from apoptosis. It is also of
interest to note that the sphingosine-induced DNA fragmentation was
observed within 6 h after the addition of sphingosine (data not shown),
while the ISP-1-induced DNA fragmentation was delayed requiring more
than 32 h to occur (Fig. 2B). These results suggest
that these two types of apoptosis caused by unusually low and high
intracellular levels of sphingolipids took place through distinct
biochemical pathways, respectively. The ISP-1-induced apoptosis seemed
to occur following one or more cell divisions (data not shown). This
type of apoptosis was also reported for hematopoietic cell lines that
had been exposed to an equitoxic dose of -irradiation and was
designated as ``delayed mitotic
death''(25, 26) .
ISP-1 inhibits the
proliferation of CTLL-2 cells and mouse splenic lymphocytes induced by
allogenic antigen stimulation(13) . In both cases IL-2 is
responsible for their proliferation as the major growth factor. These
results prompted us to suspect a direct linkage between the signal
transduction through the IL-2 receptor and the function of ISP-1. In
order to test this hypothesis, the effect of ISP-1 on the apoptosis of
another IL-2-dependent cell line, F7, which was derived from BAF-B03
cells, and acquired IL-2 dependence through the introduction of mouse
IL-2 receptor subunit cDNA (16) was studied. However,
unlike CTLL-2 cells, F7 cells did not show apoptosis in response to
ISP-1, although a high concentration of sphingosine induced the
apoptosis of F7 cells (see Fig. 4C). Furthermore, when
CTLL-2 cells were cultured in the presence IL-4 instead of IL-2, ISP-1
also induced the apoptosis of CTLL-2 cells (data not shown). These
results indicated that there is no one-to-one direct relationship
between the signal transduction through the IL-2 receptor and the
function of ISP-1. However, the possibility that F7 cells have a
specific ability to incorporate sphingolipids from the fetal calf serum
in the culture medium could not be ruled out. Since the proliferation
of stimulated T-lymphocytes was inhibited by ISP-1(14) , we
then tried to identify the type of cells that is susceptible to ISP-1.
When mouse splenic T cells were stimulated with concanavalin A in the
presence or absence of ISP-1, CD4-negative and CD8-positive cells were
much more sensitive to ISP-1 than CD4-positive and CD8-negative cells
(data not shown). Taken together, ISP-1-induced apoptosis is not
specific for IL-2-dependent cells but may be a characteristic of a
CD8-positive and CD4-negative cytotoxic T cell lineage. In sharp
contrast to the cell type-specific expression of ISP-1-induced
apoptosis, sphingosine-induced apoptosis is observed in many cell
types, including Chinese hamster ovary cells(27) , human
neutrophils (11) , and HL-60 cells(12) , as well as the
CTLL-2 and F7 cells examined in this study. The effect of sphingosine
may be associated with the protein kinase C inhibitory activity of
sphingosine(27) , because dimethylsphingosine, which is also a
protein kinase C inhibitor, induced the apoptosis of CTLL-2 cells (data
not shown), as reported in human neutrophils (11) and HL-60
cells(12) . However, the detail mechanism of the involvement of
protein kinase C in the sphingosine-induced apoptosis remains to be
elucidated.
Figure 4:
Flow
cytometry analysis of propidium iodide-stained F7 cells. Cells were
cultured with the methanol vehicle (A), 47 nM ISP-1 (B), or 5 µM sphingosine (C) for 48 h (A and B) or 8 h (C). After treatment, the
cells were analyzed as described under ``Experimental
Procedures.'' Bar 1, apoptotic cells; bar 2,
G/G
phase cells; bar 3, S phase cells; bar 4, G
/M phase
cells.