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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 8, 2000 as doi:10.1096/fj.00-0466fje. |
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,2
* INSERM E9910, Institut Claudius Régaud, 20 rue du Pont St Pierre, 31052 Toulouse, and
INSERM U466, Laboratoire de Biochimie Médicale, CHU Rangueil, 1 avenue Jean Poulhès, 31403 Toulouse, France.
The first two authors contributed equally to this work.
2Correspondence: INSERM U466, Laboratoire de Biochimie Médicale, CHU Rangueil, 1 avenue Jean Poulhès, 31403 Toulouse, France. E-mail: levade{at}rangueil.inserm.fr
SPECIFIC AIMS
The present study re-examines the controversial role of the acid lysosomal sphingomyelinase (SMase) in stress-induced apoptosis. We investigated the sensitivity of a series of acid SMase-deficient lymphoid cell lines derived from Niemann-Pick disease (NPD) patients, as well as a corrected cell line (after retrovirus-mediated gene transfer of the acidic SMase cDNA) to anthracyclines, ionizing radiation, and anti-Fas antibody.
PRINCIPAL FINDINGS
1. Lysosomal sphingomyelin turnover is deficient in NPD cells
Because controversial observations have been reported on the
behavior of NPD lymphoid cells towards stress agents, particularly Fas
ligation, the response of a series of NPD cell lines to various
inducers of apoptosis was analyzed. The panel of NPD cells we used
included the MS1418 cell line in which opposite findings have been
found. In proving that this particular cell line was indeed deficient
in acid SMase, we investigated the turnover of lysosomal sphingomyelin
by using a procedure that ensures lysosomal targeting of the
radiolabeled lipid substrate. MS1418 cells, as well as all other NPD
cell lines tested here, were indeed deficient in lysosomal
sphingomyelin catabolism, whereas its acid SMase-transduced
counterparts (line MS1418+) were fully corrected.
2. Anthracyclines induce apoptosis, caspase-3 activation, and
ceramide generation similarly in normal and NPD cells
The anticancer drugs daunorubicin and doxorubicin have been
reported to induce apoptosis, which is preceded by an increase in
intracellular ceramide levels. Whereas late (>1–3 h) ceramide
elevation is independent of SMase stimulation (due, rather, to enhanced
de novo synthesis of ceramide, because of its sensitivity to
the ceramide synthase inhibitor fumonisin B1), early ceramide
production is linked to SMase activation and subsequent sphingomyelin
hydrolysis. Because acid SMase has been described as being implicated
in doxorubicin-induced cell death, we investigated the sensitivity of
NPD cells to anthracyclines. Doxorubicin or daunorubicin treatment led
to a dose- and time-dependent cytotoxicity, which was observed in both
control and NPD lymphoid cell lines (Fig. 1A
) and due to apoptotic cell death (Fig. 1B
).
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Because current evidence suggests that anthracyclines induce apoptosis
via activation of the executioner caspases, we determined whether
treatment with daunorubicin resulted in activation of caspase-3, which
drives the effector phase of apoptosis. Daunorubicin activated
caspase-3 in control cells. Similarly, proteolytic processing of
caspase-3 was detected in NPD cells (Fig. 1C
), which
suggests that caspase-3 was comparably activated in both control and
NPD cells. We also found significant cleavage of the PARP protein, a
target of caspase-3, in control, NPD, untransduced NPD, and acid
SMase-transduced NPD cell extracts (Fig. 1D
).
Inasmuch as ceramide generation triggered by anthracyclines has been
linked to the activation of apoptosis, we sought to determine whether
the apoptosis of NPD cells induced by these agents was accompanied by
the stimulation of the ceramide pathway. This possibility was studied
by measuring sphingomyelin breakdown, ceramide generation, and SMase
activity. Much like in leukemic cells, daunorubicin treatment shortly
activated the sphingomyelin-ceramide pathway similarly in NPD and
normal lymphoid cells. Indeed, incubation with daunorubicin resulted in
a 30% increase in cellular ceramide levels, coupled with 
20%
reduction in intracellular sphingomyelin levels in both control and NPD
cells. The increase in ceramide levels was accompanied with a 30%
increase in neutral SMase activity. As expected, acid SMase activity
was not significantly affected after treatment with the anthracyclines.
3. Normal and NPD cells are equally sensitive to
irradiation-induced apoptosis
Because NPD cells were as susceptible as control cells to
anthracycline-mediated cell death, we next investigated whether acid
SMase is required for irradiation-induced apoptosis and ceramide
generation. The cytotoxicity induced by ionizing radiation (10 Gy) was
alike in control, NPD, untransduced NPD, and acid SMase-transduced NPD
cells. Indeed, the growth of control, NPD, or NPD corrected cells was
similarly inhibited. Furthermore, morphological evaluation of
irradiated cells exhibited the archetypal apoptotic features in both
normal and NPD cell types. Quantitation of these morphological changes
detected by DAPI staining of normal and NPD lymphoid cells 24 h
after irradiation indicated that NPD lymphoid cells underwent
apoptosis, in a similar fashion as their normal counterparts, to an
extent of 40%–50% apoptotic cells.
In irradiated normal lymphoblasts, increases in ceramide levels occurred rapidly and transiently within 30 min post-treatment, but also later with a more sustained elevation occurring over a period of hours after treatment. A similar pattern was found in NPD cells. In addition, a 30% increase in neutral SMase activity was observed in control and NPD cells 5–10 min post-irradiation.
4. Anti-Fas antibody induces apoptosis and caspase-3 activation in
a similar fashion in normal and NPD cells
Because the role of acid SMase in anti-Fas-triggered cell death
has been disputed, we compared the sensitivity of NPD lymphoid cells by
using an agonistic anti-Fas antibody CH-11 identical to the one used in
other studies. Treatment with anti-Fas antibody induced apoptosis
equally well in control and in NPD lymphoid cells in a dose- and
time-dependent manner. Typical apoptotic features were visible in both
normal and NPD cells treated with 200 ng/ml anti-Fas for 5 h, or
20–50 ng/ml for 5 h.
We also examined whether acid SMase influenced processing of
executioner caspases that are activated upon Fas triggering in lymphoid
cells. To determine executioner caspase activity, we used the
fluorogenic substrate, Ac-DEVD-AMC, which corresponds to the cleavage
site found in numerous caspase-3 and -7 targets. In control, NPD, and
corrected NPD cells, anti-Fas treatment resulted in a time-dependent
increase in DEVDase activity, which correlated with the onset of
apoptosis (Fig. 2A
). Proteolytic processing of caspase-3 was also examined by
Western blotting. Treatment with anti-Fas resulted in processing of
caspase-3 into its respective active forms regardless of the acid SMase
status of the cell lines used (Fig. 2B
). Although subtle
variations were found in the extent or onset of caspase activation, no
consistent differences were seen between various normal and NPD Type A
or Type B cells (up to 7 different NPD cell lines were tested). These
findings clearly demonstrate that normal and NPD cells are equally
sensitive to anti-Fas-induced apoptosis.
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CONCLUSIONS AND SIGNIFICANCE
A major interest and intense debate in sphingolipid signal transduction, and especially in apoptosis signaling, basically relies on the identification of the SMase implicated in ceramide generation. Despite a number of studies during the past years, the physiological importance of acid SMase in stress-induced apoptosis remains controversial.
Several studies have reported the implication of an acid SMase in signaling, and notably in signaling of apoptosis triggered by anti-Fas, irradiation or anthracyclines. This tenet most often relied on the resistance or an abnormal response to apoptotic stresses found by some authors in the cultured NPD cell line MS1418. However, by using the same cell line, other groups have reported a normal apoptotic response, and this finding is documented further in the present study. The differences in these responses are likely not due to phenotypic changes that may have occurred upon continuous culture, because we show here that the MS1418 cells are indeed deficient in lysosomal sphingomyelin hydrolysis. Moreover, we clearly demonstrate that different NPD cell lines all responded to diverse stress stimuli by undergoing apoptosis.
Consistent with these findings, recent studies on cells isolated from acid SMase-deficient mice indicated that lysosomal SMase is not involved in Fas-induced apoptosis of thymocytes and T and B lymphocytes. Fas engagement of activated B cells from the deficient mice also resulted in ceramide production. Moreover, apoptosis initiated by TNF has recently been reported to be unaltered in splenocytes from acid SMase-deficient mice.
Conflicting reports on the apoptotic response of NPD cells
continue. However, our laboratory in this and in other studies with
human myeloid, lymphoid, and fibroblast cells treated with
stress-inducers, such as daunorubicin, TNF, anti-CD40, and irradiation,
has observed only the activation of a neutral SMase as have others
using similar cell models and other effectors. Therefore, the present
study strongly suggests that the contribution of the lysosomal acid
SMase appears to be limited (Fig. 3
) and that other SMases await purification and cloning, as well as
animal studies. That acid SMase knockout mice partially resist
hepatocyte apoptosis after injection of anti-Fas does not definitely
prove the role of lysosomal SMase in apoptosis signaling. The acid
SMase defect may indirectly affect the plasma membrane composition, and
as a result alter signaling processes. Resistance to liver injury was
seen only for low doses of anti-Fas, which was partially overcome
with slightly higher doses. Additional studies are needed to assess the
role of the general lipid storage and abnormal ultrastructure of liver
cells in this relative resistance.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0466fje To cite this
article, use (December 8, 2000) FASEB J. 10.1096/fj.00-0466fje 
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