From the Life Science Group, During the exposure of human myelocytic leukemia
HL-60 cells to phorbol diester, nonadherent cells die by apoptosis, but
adherent cells survive and growth-arrest at G1 phase
of the cell cycle. Here we have shown that the adherent cells rapidly
died by apoptosis after forced detachment (anoikis), indicating that
phorbol diester induced apoptosis by default. Dimethylsphingosine
induced apoptosis in the adherent cells, and sphingosine-1-phosphate
rescued the detached cells from apoptosis. Sphingosine kinase activity
in adherent cells was higher than that in nonadherent cells and was decreased by forced detachment. It is likely that the phorbol diester-induced apoptosis and the adhesion-mediated survival are modulated by sphingosine and sphingosine-1-phosphate, respectively. The
adherent cells were reverted and reproliferated when allowed to
spontaneously detach from plastic surfaces by removal of phorbol diester. This result suggests that after removal of phorbol diester, the commitment signal of apoptosis by default is lost faster than the
survival signal by adherence.
In phorbol ester-induced monocyte/macrophage-like differentiation
in human leukemic HL-60 cells (1), the leukocyte integrin receptor
Mac-1 is expressed on the cell surface (2), and the cells are
growth-arrested at G1 of the cell cycle (3, 4). Mac-1 (CR3,
Phorbol esters also induce apoptosis of HL-60 and U937 cells (21),
which is accompanied by a marked decrease in bcl-2 mRNA and protein levels (22). Ectopic expression of Bcl-2 protein inhibits
apoptosis but has no significant effect on differentiation (23, 24).
Apoptosis of HL-60 cells induced by treatment with phorbol esters has
been correlated with an increase in the steady-state level of
sphingosine and elevation of the ceramidase activity (25). In fact,
exposure of HL-60 cells to sphingosine induced apoptosis (25, 26).
Co-exposure of HL-60 cells to phorbol ester and sphingosine blocked
phorbol ester-induced differentiation (27, 28). Interestingly, the cell
adherence to plastic substrate protected HL-60 cells from apoptosis
induced with phorbol esters (29). Recently, sphingosine-1-phosphate
(SPP)1 has been shown to
prevent apoptosis in HL-60 cells or U937 cells induced by tumor
necrosis factor- The several lines of experimental evidence summarized above
suggested to us the following possibilities. 1) Apoptosis by
sphingosine occurs by default (31) in phorbol ester-treated HL-60
cells; 2) adherence to plastic by Mac-1 elicits an
anchorage-dependent survival signal, and adherent cells may
undergo anoikis by forced suspension (32, 33); 3) apoptosis in
nonadherent cells and anoikis, as well, may be suppressed by the
addition of SPP; and 4) if the commitment signal to apoptosis is lost
faster than the survival signal, phorbol ester-differentiated and
adherent HL-60 cells may de-differentiate and proliferate again by
removing phorbol ester without perturbing the adherent state. Here we
demonstrate that our model is substantiated by a line of experimental
evidence that was obtained by studying adherent and suspended cell
fractions separately after isolation.
Cell Culture and Drug Treatment
The human acute myeloblastic leukemia cell line HL-60 (34-36)
(CCL240; ATCC, Manassas, VA) was cultured in plastic dishes at 37 °C
in RPMI 1640 medium (Nissui, Tokyo, Japan) supplemented with 10% fetal
calf serum (FCS, Irvine Scientific, Santa Ana, CA) and 100 µg/ml
kanamycin. The HL-60 cells used in this work were restricted to early
passages characterized by a doubling time of 47 h. Cells were
passaged before cell density reached 2 × 106 cells/ml
and seeded at 3 × 105 cells/ml. The cell viability
determined by trypan blue exclusion was scored during the course of
culture and found to be higher than 90%. For the differentiation
induction we used phorbol 12,13-dibutyrate (PDB; Sigma) rather than
phorbol 12-myristate-13-acetate (PMA), because PDB can be easily
removed from cells by simple washings (37), provided that FCS or BSA is
contained in the washing medium (38). An ethanol stock solution of PDB
(0.2 mM) was diluted before use to 10 µM with
the culture medium and added to a cell culture of 5 × 105 cells/ml at a final concentration of 100 nM
(4). In using sphingolipids as effectors, cells in logarithmic growth
were first precultured for 24 h in a serum-free RPMI 1640 medium
supplemented with insulin (5 µg/ml) and transferrin (5 µg/ml) and
treated with drugs in the same medium (30, 39). An ethanol stock
solution (10 mM) of
N,N-dimethylsphingosine (DMS; Biomol) was diluted
with the serum-free medium before use. SPP (Biomol) was added as BSA complexes (39) at a concentration of 125 µM in 4 mg/ml
BSA (fatty acid-free). In suppression of anoikis of HL-60 cells with
SPP, adherent cells prepared by pretreatment with 100 nM
PDB for two days were detached by forced suspension by pipetting three
times with the serum-free medium containing 20 µM SPP.
PDB was removed from cells by washing three times with RPMI 1640 containing 5% FCS, and FCS was then removed by washing with serum-free
medium. The cells were finally suspended in serum-free medium
containing 20 µM SPP.
Separation of Adherent and Nonadherent Cell Populations
During the PDB treatment of HL-60 cells, suspended cells were
separated by gentle pipetting. Culture dishes with the adherent cells
were gently washed three times with 10 ml of fresh medium. A small
number of cells contained in the wash fractions were discarded. The
number of adherent cells were counted without suspending them from
dishes by using an inverted microscope fitted with an ocular lens with
grid, because the cells formed large aggregates after suspension. A
unit area of the grid with an appropriate size was chosen so that a
total of 200 or more adherent cells were contained in it. The cell
density/unit area was measured at 8-16 different places selected at
random in a dish. The total number of adherent cells/dish was estimated
by multiplying the average cell density and the total area of a
dish.
Assay of Cell Proliferation
To a cell culture of 100 µl (3-10 × 105
cells/ml), [3H]thymidine (55 Ci/mmol; ICN Biomedicals,
Costa Mesa, CA) was added to a final activity of 0.5 µCi/ml. After
incubation for 1 h at 37 °C in a CO2 incubator, the
cells were washed 3 times and fixed on a slide glass. Autoradiography
was done by immersing the slide glass in autoradiographic emulsion
(type NR-M2; Konica, Tokyo, Japan) and exposing it for 4-6 days.
Developed and fixed slides were counterstained with Giemsa, and more
than 400 cells were counted under a light microscope. The labeling
index was calculated from the number of cells with silver grains
divided by the total cell counts.
Assay of Apoptosis
Nuclear Fragmentation--
105 cells were fixed with
an equal volume of 5% paraformaldehyde, neutralized by one-tenth
volume of 1 M Tris-HCl (pH 7.2) and centrifuged onto a
glass slide using a cytospin apparatus. The cells were further fixed in
cold methanol ( DNA Fragmentation (40)--
Briefly, 107 cells were
lysed in a lysis buffer containing 0.5% Triton X-100, 10 mM EDTA, 10 mM Tris-HCl (pH 7.4) and
centrifuged. The supernatant containing the fragmented DNA molecules
was recovered and digested with RNase A followed by proteinase K. Nucleic acids were precipitated in 50% isopropanol and 0.5 M NaCl. The precipitates were pelleted by repeated high
speed centrifugation and used for analyses by electrophoresis on a
1.5% agarose gel and staining with ethidium bromide. The DNA migration
was assessed in an image analyzer (FAS-II, Toyobo, Osaka, Japan).
Suspension of Adherent Cells
Forced Suspension--
After separation of nonadherent cells,
adherent cells were washed four times with RPMI 1640 medium containing
5% FCS. The adherent cells were then suspended by the following three
different methods. 1) The adherent cells were incubated for 10 min in
phosphate buffered saline with no Ca2+ or Mg2+
and then suspended by pipetting; 2) the adherent cells were first overlaid with 5 ml of 0.05% trypsin, and 4.5 ml of the supernatant was
removed and incubated at 37 °C for 5 min to allow the cells to round
up. The cells were suspended by repeating the addition of RPMI 1640 containing 10% FCS and pipetting; 3) addition of RPMI 1640 containing
10% FCS and pipetting were repeated several times until the cells were
completely suspended. Culture of the suspended cells was started by
seeding at 5 × 105 cells/ml in normal medium of RPMI
1640 containing 10% FCS.
Spontaneous Suspension--
After the treatment of HL-60 cells
with 100 nM PDB for 2 days, nonadherent cells were
separated by gentle pipetting. PDB was then removed by a procedure that
consisted of 1) medium changes for 3 times with fresh medium containing
5% FCS, 2) incubation at 37 °C for 1 h in a CO2
incubator, and 3) further medium changes for 3 times. The medium change
was made as gently as possible such that the cell adherence was not
disturbed. During further incubation in fresh medium, the adherent
cells spontaneously detached from plastic surfaces.
Sphingosine Kinase Assay
Sphingosine kinase activity was measured essentially as
described previously (41). Briefly, 107 cells were lysed by
freeze-thawing in 0.2 ml of 0.1 M phosphate buffer (pH 7.2)
containing 10 mM MgCl2, 20% glycerol, 1 mM mercaptoethanol, 1 mM EDTA, 20 µM ZnCl2, 1 mM
Na3VO4, 15 mM NaF, 10 µg/ml
leupeptin and aprotinin, 1 mM phenylmethylsulfonyl
fluoride, and 0.5 mM 4-deoxypyrodoxine. Cytosolic fractions
were prepared by ultracentrifugation at 105,000 ×g for 90 min. Sphingosine kinase activity in supernatants (50 µl) was measured
by incubating with 5 µM sphingosine-BSA complex (42) and
[ Plastic Adherence and Cessation of Proliferation by Exposure to
PDB--
PDB-treated HL-60 cells followed different fates. The number
of differentiated cells that adhered to plastic surfaces began to
increase after an induction period of at least 6 h (4) and reached
a plateau in 24 h (Fig.
1A, closed
circles). We showed by using an antisense RNA technology that the
plastic adherence was mediated by Mac-1 expressed at the cell
surface.2 The adherent cells
were growth-arrested (Fig. 1B, closed circles) at
the G1 phase of the cell cycle. A small number of adherent cells in the induction period were spontaneously differentiated cells
(44). The number of undifferentiated, nonadherent cells exhibited an
inverse decrease and leveled off in 24 h (Fig. 1A, open circles). The remaining nonadherent cells were
apoptotic and appeared resistant to adherence. In the early stage of
PDB treatment, the nonadherent cells continued to proliferate, although at a progressively reduced rate (Fig. 1B, open
circles). Most of the nonadherent cells were converted to adherent
cells by induction of differentiation; the remaining cells either
proliferated by self renewal in the early stage or underwent apoptosis
in the later stage and were gradually degraded (see below). Therefore, we plotted in Fig. 1A the number of cells for each type in a
dish rather than the percentage of total.
Helix Research Institute,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
M
2, CD11b/CD18) is a member of the
2 integrin subfamily and plays a critical role in
numerous physiological functions of monocytes and macrophages that are
mediated by cell-cell and cell-substrate interactions (5-8, 46).
Phorbol esters activate Mac-1 receptors on monocyte such that they
promote vigorous phagocytosis (9, 10). Monocytes die by apoptosis after
phagocytosis of bacteria (11). Undifferentiated HL-60 and U937 cells
express LFA-1 (
L
2, CD11a/CD18) (2),
another member of the
2 integrin subfamily, although at
low levels. CD18 subunit mRNA is thus expressed constitutively, but
CD11b subunit mRNA is not expressed at detectable levels (12).
Phorbol ester up-regulates the steady state levels of both CD11b
mRNA (13) and CD18 mRNA (14, 15) by transcriptional activation.
Firm adherence of differentiated cells to tissue culture plastic dishes
is inhibited by a monoclonal antibody to CD18 (16) or CD11b (17).
Transcription factor NF-
B is constitutively activated by phorbol
esters (18) and is indispensable for the CD11b gene
expression and cell adhesion, because both are suppressed by a dominant
negative inhibitor of NF-
B expression (19) and by antisense
oligonucleotides to RelA subunit (20).
(TNF-
) or Fas ligand (30). The principal
mediator of apoptosis in these cases is ceramide, not sphingosine, but
SPP could be equally effective in the protection of adherent HL-60
cells from phorbol ester-induced apoptosis.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
20 °C, 5 min) followed by cold acetone (
20 °C,
5 min), and the plates were allowed to dry. Dried plates were stained
with 2 µg/ml 4,6-diamidino-2-phenylindole (DAPI). Alternatively,
cytospin preparations were fixed in 90% (v/v) cold methanol
(
20 °C, 5 min) and dried. Dried plates were stained with 125 µg/ml acridine orange in phosphate buffer (pH 6.9). Nuclei were
assessed in an Olympus OMT2 inverted fluorescence microscope equipped
with the appropriate epifluorescence filters at a final magnification
of 1500×.
-32P]ATP (1 mM, 0.2 Ci/mmol) for 30 min
at 37 °C. Labeled lipids were extracted with a mixture of
chloroform/methanol/concentrated HCl (100:200:1, by volume), and the
extract was partitioned into two phases by adding 2 M
KCl/methanol (1:1, v/v) (43). The lower phase was further washed twice
with the same mixture and dried in a vacuum centrifuge at room
temperature. The dried lipid was completely dissolved in 100 µl of
chloroform/methanol (1:1, v/v) with occasional stirring over 30 min.
Lipids were resolved on silica gel plates (Silica Gel 60; Merck)
impregnated with 1% potassium oxalate/2 mM EDTA, using the
solvent system of butanol/water/acetic acid (3:1:1) (39). The
phospholipid standards were visualized with molybdenum blue spray
(Sigma), and the radioactivity was measured by autoradiography in a
Bioimaging analyzer (BAS2000, Fuji Film, Tokyo, Japan).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (15K):
[in a new window]
Fig. 1.
Induction of differentiation and growth
arrest of HL-60 cells by exposure to 100 nM PDB.
A, the number of adherent cells (closed circles)
and suspended cells (open circles) during the PDB treatment
in 10-cm culture dishes. B, the labeling indices for the
adherent cells (closed circles) and the nonadherent cells
(open circles). TdR, thymidine.
Survival of Adherent Cells and Apoptosis of Nonadherent Cells During Exposure to PDB-- Most of the nonadherent cells exhibited an apoptotic morphology of nuclear fragmentation between day 1 and day 2 of PDB treatment, but no nuclear fragmentation was observed in adherent cells (Fig. 2A). However, nuclear fragmentation occurred rapidly when the adherent cells were forced to suspend in medium that contained no PDB, independently of the method of suspension (Fig. 2B). This result indicates that PDB-treated adherent cells have already been committed to apoptosis (apoptosis by default). In the observed anoikis, apoptotic cell death by detachment, the apoptotic cells were later degraded and lost from the system, as indicated by the decrease in the percentage of cells with nuclear fragmentation after 12 h (Fig. 2B). The internucleosomal DNA fragmentation was consistent with the observed nuclear fragmentation. DNA fragmentation was demonstrated in nonadherent cells (Fig. 2C, lanes 4-6), whereas no fragmentation was observed in adherent cells (Fig. 2C, lanes 7-9). However, DNA fragmentation did occur in adherent cells after the forced suspension (Fig. 2C, lanes 12-13). The adherent cells did not survive long term, as they gradually detached from the plastic surface after 3 days and later died by apoptosis. Similar results were reported previously (29).
|
Suppression of PDB-induced Apoptosis in HL-60 Cells by
SPP--
SPP was investigated for its ability to block apoptosis in
HL-60 cells induced by PDB, as it has been shown to block the apoptosis by TNF- or Fas ligand (30). Apoptosis induced by PDB treatment of
the nonadherent cells in either serum-free RPMI 1640 or medium supplemented with 10% FCS progressed similarly, as observed by nuclear
fragmentation (Fig. 3A,
open bars). SPP at 20 µM strongly inhibited
the apoptosis at day 2, but the inhibitory effects were almost gone by
day 3 (Fig. 3A, hatched bars). Anoikis induced by
forced suspension of PDB-treated adherent cells was suppressed by SPP
beyond the experimental error (Fig. 3B), although the extent of inhibition was less than in nonadherent cells (Fig. 3A).
Since the anoikis progressed very rapidly after the forced suspension compared with the apoptosis of PDB-treated nonadherent cells, we tried
to add 20 µM of SPP to the medium 5 h before the
detachment, but it failed to protect cells from anoikis.
|
Induction of Apoptosis of HL-60 Cells by DMS and Its Suppression by SPP-- We further examined the involvement of sphingosine in PDB-induced apoptosis of HL-60 cells and suppression of apoptosis by SPP. HL-60 cells were incubated with DMS, a strong inhibitor of sphingosine kinase, for 12 h in serum-free RPMI 1640 medium, and nuclear fragmentation was observed by staining with DAPI (Fig. 4A). The percentage of cells with fragmented nuclei increased in a dose-dependent manner. A similar result was reported previously by observation of DNA fragmentation (25, 26). The apoptosis induced by DMS was inhibited in a dose-dependent manner by the simultaneous addition of SPP (Fig. 4B). HL-60 cells remained suspended throughout these treatments. The differentiated, adherent HL-60 cells induced by the PDB treatment for 12 h also underwent apoptosis by the addition of DMS (Fig. 4C) to the same extent as did the untreated HL-60 cells (data not shown). The PDB-differentiated HL-60 cells adhered to the plastic surface by spreading pseudopodia-like structures so firmly that the cells were only suspended by vigorous pipetting. The HL-60 cells co-treated with PDB and DMS still adhered to the plastic surface, and the morphology did not change appreciably from the PDB-differentiated cells, but the cells were suspended very easily by light pipetting. Apoptosis of HL-60 cells was not induced by the treatment with 10 µM of C2-ceramide for 24 h, in agreement with previous results (25) (data not shown).
|
Activation of Sphingosine Kinase by Adherence of PDB-treated HL-60 Cells-- Sphingosine kinase activity was measured for adherent and nonadherent cell fractions after the PDB treatment (100 nM) for 15 h. The percentage of adherent cells was 44 ± 3%. Fig. 5A shows that the activity was higher in both the adherent and nonadherent cells compared with the untreated cells, but the value for the adherent cells was much higher than that for the nonadherent cells. The detached cells by forced suspension also exhibited a decreased sphingosine kinase activity compared with the adherent cells (time 0) after further incubation for 4 h in the absence of PDB (Fig. 5B). The addition of PDB during the further incubation increased the activity only slightly. The sphingosine kinase activity for the detached cells at 8 h after suspension was not significantly different from the value for the adherent cells both in the presence or absence of PDB.
|
Survival from Apoptosis and Reproliferation of Adherent Cells after Spontaneous Suspension-- After the treatment of HL-60 cells with 100 nM PDB for 2 days, the cells that adhered to 10-cm plastic dishes were separated by gentle pipetting of the nonadherent cells. The medium was gently replaced by the fresh medium containing no PDB such that the cell adherence was not disturbed. During further incubation in fresh medium not containing PDB, the adherent cells spontaneously detached, as shown by the decrease in the number of adherent cells (Fig. 6A). The detached cells were collected during various time intervals after the removal of PDB, and the time course of apoptosis was examined for each fraction after suspension. Quite interestingly, those cells that adhered longer exhibited less apoptosis as assayed by the nuclear morphology (Fig. 6B). Those cells that spontaneously detached after 24 h showed essentially no apoptosis (Fig. 6B, closed circles). The HL-60 cells that spontaneously detached after 12 or 24 h did not clearly exhibit apoptosis as observed by internucleosomal DNA fragmentation (Fig. 2C, lanes 10-11). The percentage of apoptotic cells decreased with time (Fig. 6B) due to both the degradation of apoptotic cells and the reproliferation of suspended cells (Fig. 6C). It is interesting to note that those detached cells that suspended earlier after the removal of PDB more rapidly entered S phase in the fresh medium, a correlation inconsistent with cell death by apoptosis. All the [3H]thymidine-labeled cells were co-labeled by immunostaining with monoclonal antibody to Mac-1, indicating that the reproliferating cells were those cells that had been differentiated by the previous PDB treatment.3 These results indicate that the commitment of apoptosis by default is canceled by PDB removal before the cells lose the anchorage-dependent survival signal, resulting in de-differentiation and reproliferation of the differentiated cells.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anoikis of PDB-differentiated HL-60 Cells--
In the induction of
differentiation of HL-60 cells by exposure to 100 nM PDB,
most cells firmly adhered to the plastic surface within 24 h (Fig.
1A), stopped proliferating, and arrested at G1
of the cell cycle (Figs. 1B, 2A, and
2C). The PDB concentration of 100 nM is thus not
severely cytotoxic and can be used for the study of differentiation
induction. The persistently nonadherent cells (Fig. 2A) died
by apoptosis after 24 h (Fig. 2C). Similar results were
reported previously (29). Differentiation as observed by Mac-1-mediated
adherence was induced before apoptosis. The studies with a HL-60 cell
line with overexpressed bcl-2 gene suggest that
differentiation is regulated independently of apoptosis (23, 24).
It is interesting to note in this regard that high passage HL-60
sublines, which have amplified sequences in a single homogeneously staining region in a chromosome, were resistant to both differentiation and apoptosis after PDB
treatment.4 The HL-60 cells
studied here were of low passage and have double minutes but no
homogeneously staining region. Amplified sequences of genes such as
c-myc are detected in both the homogeneously staining region
and double minutes (45), but the genomic contents may not necessarily
be identical with each
other.5 Therefore,
differentiation or the differentiation potential could be a
prerequisite for apoptosis. Expression of 2 integrin
Mac-1 on the cell surface was lost from the cells en route to
apoptosis, but it was retained in the reproliferating cells after
removal of PDB.3
The involvement of Sphingolipid Metabolites in the Regulation of
Apoptosis or Survival in PDB-treated HL-60 Cells--
It has recently
been suggested that the block by PMA of apoptosis induced by TNF- in
HL-60 cells and U937 cells is mediated by activation of
sphingosine-1-kinase and the resultant increase in SPP levels (30).
Activation of transcription by NF-
B (50-52) could be an important
upstream event. Intriguingly, PMA increased both the SPP and
sphingosine levels in the presence or absence of TNF-
(30). TNF-
increased the cellular concentration of ceramide, but TNF-
had no
effect on the amount of basal sphingosine. The involvement of ceramide
in TNF-
-induced apoptosis has been questioned, however, by a direct
and simultaneous determination of sphingolipids using a mass
spectrometric technique, that showed no generation of ceramide by
TNF-
(53). In contrast to TNF-
, PMA increased sphingosine levels
but did not increase basal levels of ceramide, even in the presence of
TNF-
. It was reported that C2-ceramide (10 µM) induced apoptosis in HL-60 cells, but we could not
detect significant apoptosis in agreement with other reports (25, 26,
54). The reason for the discrepancy is presently unknown.
Reversion of PDB-differentiated HL-60 Cells-- We showed that PDB-differentiated, adherent cells reproliferate if PDB is removed from the culture medium without disturbing the adherent state (Fig. 6C). This result implies that the commitment to apoptosis by default induced by PDB is resolved faster than the loss of survival signal by adherence after the PDB removal. Those cells that adhered longer to the culture dish after the PDB removal exhibited less apoptosis after the spontaneous detachment (Fig. 6B). We have shown recently that the irreversible differentiation of HL-60 cells by exposure to dimethyl sulfoxide is induced by elimination of double minutes that are trapped in micronuclei.5 It is interesting to note in this regard that PDB inhibited micronucleation and elimination of double minutes. Therefore, integrity of the genomic constituents in HL-60 cells is maintained after the PDB treatment.5 Thus there may exist a reasonable route of de-differentiation of PDB-differentiated HL-60 cells as demonstrated in this study. Previously, the reproliferation of PMA-treated U937 cells after long term culture was referred to as retrodifferentiation just to indicate that the process was not a simple reversion (60, 61). However, long term culture may be unnecessary if anoikis is effectively prevented.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. S. Ikegami, H. Matsuda, and K. Akahori for stimulating discussions and Dr. N. Fukamiya for TLC experiments.
![]() |
FOOTNOTES |
---|
* This work has been supported by grants from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 81 82 424 6561; Fax: 81 82 424 0759.
1
The abbreviations used are: SPP,
sphingosine-1-phosphate; TNF-, tumor necrosis factor-
; FCS, fetal
calf serum; PDB, phorbol 12,13-dibutyrate; PMA, phorbol
12-myristate-13-acetate; DMS,
N,N-dimethylsphingosine; BSA, bovine serum
albumin; DAPI, 4,6-diamidino-2-phenylindole.
2 Hamada, K., Nakamura, H., Oda, T., Hirano, T., Shimizu, N., and Utiyama, H. (1998) Biochem. Biophys. Res. Commun. 244, 745-750
3 T. Oda and H. Utiyama, H., unpublished observations.
4 H. Nakamura and H. Utiyama, unpublished observations.
5 K. Kitajima, H. Nakamura, T. Hirano, K. Hamada, M. Haque, N. Itoh, H. Shimokawa, K. Tanaka, N. Kamada, N. Shimizu, N., and H. Utiyama, submitted for publication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|