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Research Communications |
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: programmed cell death • protection • growth factors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B has
also been shown to play a role in altering glucocorticoid action,
suggesting that molecules from other signaling pathways are capable of
regulating the effect of glucocorticoids. Many chemotherapeutic agents,
such as glucocorticoids, methotrexate, vincristine, and cisplatin, are
known to inhibit cell growth and induce apoptosis; however, the
efficacy of such chemotherapeutic agents is often modified by the Bcl-2
oncogene (1
, 2
). Thus, analysis of the Bcl-2
expressing status of a tumor may be useful in selecting
chemotherapeutic strategies for cancer therapy.
The Bcl-2 gene is a repressor of apoptosis that is homologous to the
Caenorhabditis elegans ced-9 gene (3)
. This
gene was first identified at the breakpoint of t (14 : 18) (q 32 ; 21)
of a human follicular B cell lymphoma (4
, 5
).
There are many gene products in the Bcl-2 multigene family
(6)
. Interaction between some members of this family is
capable of modulating apoptosis. Overexpression of Bcl-2 inhibits
apoptosis induced by a wide variety of stimuli such as glucocorticoids
(7)
, anti-Fas antibody (8)
, radiation
(9)
, tumor necrosis factor (TNF)-
(10)
,
and sphingolipids (11)
, but Bcl-2 does not suppress
apoptosis induced by all signals (12
, 13
).
Although the mode of Bcl-2 action is poorly understood, inhibition of
the interleukin-1ß converting enzyme (ICE)3
in protease
cascade in response to an apoptotic agent has recently been implicated
in its mechanism (14)
. Caspases have received considerable
attention and are believed to play a crucial role in the upstream
signaling pathway of the apoptotic machinery. The requirement for
cytochrome c for activation of caspase-3-like activity has
also been shown (15)
. Binding of Bcl-2 to Bax has been
proposed to block the function of Bax 16-19)
, a
channel-like protein potentially responsible for releasing cytochrome
c from the mitochondria during caspase-3 activation. In
contrast to alteration of Bax function, the conversion of Bcl-2 protein
to a Bax-like protein via cleavage of Bcl-2 protein by caspases has
also been proposed recently (20)
. Moreover, inhibition of
caspase activation by indirect binding of Bcl-2 protein via a
ced-4-like binding protein has been suggested (21
,
22
).
To examine potential relationships between the Bcl-2 and the glucocorticoid signaling pathways, we studied the growth inhibition and apoptosis-inducing effects of glucocorticoids and serum depletion on S49 mouse T lymphoma cells, which are sensitive to glucocorticoid-induced apoptosis and growth inhibition. In contrast, S49 cells overexpressing Bcl-2 (S49 Bcl-2) are resistant to glucocorticoid-induced apoptosis, but not to glucocorticoid-inhibited cell growth (7) . We have now assessed the effects of serum depletion on growth inhibition and apoptosis induction in the presence of several inducers of apoptosis as a function of Bcl-2 expression. We demonstrate that although all apoptotic inducers inhibited cell growth, glucocorticoids and protein synthesis inhibitors were capable of inhibiting apoptosis induced by serum depletion in Bcl-2 overexpressing cells. These results suggest that expression of Bcl-2 can switch glucocorticoids from a proapoptotic to an antiapoptotic agent, probably via mechanisms that involve inhibition of protein synthesis (23) .
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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phage DNA,
and the Hae III digest of 
X 174 were purchased from GibcoBRL
(Gaithersburg, Md.). Fetal calf serum (FCS) was obtained from Summit
Biotechnology (Fort Collins, Colo.). L-Glutamine, propidium iodide,
ethidium bromide, DL-dithiothreitol (DTT), dimethyl sulfoxide (DMSO),
N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] (HEPES),
3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate
(CHAPS), nonidet P-40 (NP-40), Triton X-100, thapsigargin, and
anisomycin were purchased from Sigma Chemical Co. (St. Louis, Mo.).
Dexamethasone was obtained from Steraloids (Wilton, Ohio). Penicillin,
streptomycin, and proteinase K were obtained from Boehringer Mannheim
(Indianapolis, Ind.). Ethanol was obtained from Pharmco Products, Inc.
(Brookfield, Conn.). Tris and magnesium chloride were purchased from
Fisher Scientific (Houston, Tex.). EDTA was obtained from EM Science,
Curtin Metheson Scientific (Houston, Tex.). Phenol was purchased from
United States Biochemical (Cleveland, Ohio). Chloroform was obtained
from Mallinckrodt Chemicals (Paris, Ky.). Isoamyl alcohol was purchased
from J. C. Baker, Inc. (Phillipsburg, N.J.). Anti-Fas antibody was
bought from PharMingen (San Diego, Calif.). Cycloheximide was bought
from Calbiochem-Novabiochem Co. (La Jolla, Calif.). Sodium hydroxide
was obtained from Mallinckrodt Specialty Chemicals Co. (Paris, Ky.).
DEVD-FMK (DEVD-fluoromethylketone) and DEVD-AFC
(DEVD-7-amino-4-trifluoromethylcoumarin) were purchased from Kamiya
Biomedical Co. (Thousand Oaks, Calif.). RU 486 was a gift from Roussel
UCLAF (Romainville, France).
Cell culture
S49 Neo and S49 Bcl-2 cells were created by stable transfection
with the neomycin resistance gene alone or in combination with the
Bcl-2 proto-oncogene (1)
. The immature murine T lymphoma
cell lines S49 Neo and S49 Bcl-2 cells were cultured in RPMI-1640
medium supplemented with 10% heat-inactivated FCS, 4 mM L-glutamine,
100 I.U./ml penicillin, and 70 I.U./ml streptomycin (7)
.
The cell cultures were maintained at 1–5 x 105
cells/ml at 37°C in a 7% CO2 humidified environment and
passaged every other day. Under experimental conditions, S49 Neo and
S49 Bcl-2 cells were cultured in RPMI-1640 medium supplemented with
different concentrations (1%, 2%, 5%, 10%) of heat-inactivated FCS.
Cultures for experiments were initially plated at 2 x
105 cells/ml in a total volume of 10 ml while viability was
not less than 96%. At the end of culture, cell growth was measured by
determining cell density in a hemocytometer and viable cell number was
quantitated by trypan blue exclusion. The percent viability was
calculated by dividing the number of non-trypan blue-stained cells by
the total number of cells.
Analysis of apoptosis by flow cytometry
Flow cytometry was used to assess the mode of cell death
(24)
that occurred in response to FCS depletion or
glucocorticoid administration. S49 Neo and S49 Bcl-2 cells were
pelleted from each culture after cell counting and fixed by addition of
cold 70% ethanol with agitation. The cells were then stored at 4°C
for at least 24 h and for up to 1 wk. Fixed cells were pelleted
and washed with 1x phosphate-buffered saline (PBS), then stained with
20 µg/ml propidium iodide containing 1 mg/ml RNase in 1x PBS.
Stained cells were examined on a Becton-Dickinson FACsort using
CELLQuest software (Becton-Dickinson Immunocytometry System, San
Jose, Calif.). Individual populations of cells (10,000 per
experimental sample) were selected by gating on an area vs. width dot
plot to exclude cell debris. The data gathered from flow cytometry were
then analyzed using DeltaGraph Pro 3D software. The difference in
subdiploid peaks was examined by analyzing the average number obtained
by gating subdiploid peaks from three experiments.
Analysis of DNA integrity
DNA was prepared by pelleting ~1 x 107
cells, followed by resuspension in whole cell lysis buffer (5 mM Tris,
20 mM EDTA, and 0.5% Triton X-100, pH 8.0). Cell lysates were then
treated twice with 0.4 mg/ml proteinase K for 1 h at 55°C. The
samples were extracted twice with phenol:chloroform:isoamyl alcohol
(25:24:1) and once with chloroform:isoamyl alcohol (24:1). The DNA was
precipitated by adding NaCl to a final concentration of 0.05 M and 1 ml
ice-cold 100% ethanol, followed by incubating for 30 min at -70°C.
The precipitated DNA was pelleted at 4°C and dried 20 min in a
SpeedVac (Savant Instruments, Inc., Farmingdale, N.Y.). TE (10 mM Tris,
1 mM EDTA, pH 7.4) containing 10 mg/ml DNase-free RNase A was added to
samples and incubated overnight at 37°C. DNA concentrations were
measured by UV absorbance at 260 nm. One microgram of Hind III and
X 174 DNA marker and 15 µg of S49 Neo or S49 Bcl-2 DNA/lane were
loaded onto a 1.8% agarose gel and electrophoresed in 0.5x TPE (40 mM
Tris-phosphate, 4 mM EDTA) for 3 h, 15 min at 80 V
(25
, 26
). The gel was then stained in running
buffer containing 10 mg/ml ethidium bromide for 1 h and visualized
by UV transillumination.
Analysis of caspase-3-like activity
Caspase-3-like activity was measured by using the protocol
provided by Kamiya Biomedical Co. S49 Neo or S49 Bcl-2 cells treated
with various concentrations of dexamethasone were harvested by
pelleting 2.5 x 107 cells from cultures in 10% or
1% FCS-supplemented media. Resuspension of the pellet in nuclei
isolation buffer (10 mM MgCl2, 0.25% NP-40) was followed
by centrifugation for 30 min at 100,000 x g at 4°C,
in a Beckman Ti-100 tabletop ultracentrifuge (Beckman Instruments,
Inc., Palo Alto, Calif.). The supernatant was mixed with ICE buffer (50
mM HEPES, 10% sucrose (w/v), 0.1% CHAPS, pH 7.5), DTT, and DEVD-FMK,
which were added to 10 mM and 12.5 µM, respectively, and then
incubated for 15 min at 30°C as a nonspecific protease control.
DEVD-AFC (50 µM) was added after the incubation. A mixture of all
reagents except DEVD-FMK was prepared for measuring caspase activity.
All tubes were subsequently incubated for 5 min at 30°C, followed by
measurement of fluorescence (excitation 400 nm, emission 505 nm) in a
Perkin-Elmer 650-40 fluorescence spectrophotometer (Perkin-Elmer Co.,
Norwalk, Conn.). The samples were then incubated for another 30 min at
30°C before the second measurement of fluorescence. Each unit of
caspase activity was defined as the amount of caspase required to
produce 1 pmol of AFC/min at 25°C at saturated substrate
concentrations. AFC calibration curves were prepared with x = pmol AFC
and y = fluorescence units (FU) by measuring different dilutions of
AFC. The slope of this curve was calculated by FU/AFC and the
fluorescence change (
FU) was determined by subtracting the initial
fluorescence at time 0 from fluorescence after 30 min in the presence
of sample and substrate. Caspase activity was standardized by dividing
the units of caspase activity with total cytosolic protein. Results
were analyzed with Microsoft Excel 5.0 software (27)
.
Statistics
Paired t test was used to analyze all data collected
from cell culture showing cell number and viability, the percentage of
subdiploid peaks from flow cytometric results, and caspase-3-like
activity by comparing different treatments to control group. One-way
and two-way ANOVA (analysis of variance) were used as global tests to
examine the trend of serum depletion and dexamethasone effects. All
statistical analyses were performed in Microsoft Excel 5.0 software.
Statistical significance was defined as P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since the accumulation of viable cells reflects the corresponded effects of cell growth plus cell death, we examined the effects of glucocorticoids and serum depletion individually and in combination on cell accumulation and death in this model system. After 48 h treatment, dexamethasone inhibited S49 Neo cell growth in a dose-dependent manner in 2%, 5%, and 10% FCS-supplemented media, but significantly inhibited growth of S49 Bcl-2 cells only in 5% and 10% FCS-supplemented media (Fig. 2 A). Dexamethasone had no additional inhibitory effect on cell growth in S49 Neo cells cultured in 1% FCS-supplemented medium or S49 Bcl-2 cells cultured in 1% or 2% FCS-supplemented media. Together, these data suggest that serum depletion and glucocorticoids act convergently to inhibit cell growth in lymphocytes or that growth-inhibited lymphocytes are resistant to glucocorticoids.
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Analysis of the combined effect of serum depletion and glucocorticoids on cell viability is shown in Fig. 2B . S49 Neo cells were shown to be killed by serum depletion and dexamethasone in a dose-dependent manner. The effect of dexamethasone on cell death was additive with the effect of serum depletion (in 1% serum-supplemented medium) in contrast to the effect of these two stimuli on cell growth. S49 Bcl-2 cells were killed by serum depletion in a dose-dependent manner (Fig. 2B ), but when cultured in 5% (7) and 10% FCS-supplemented media, were resistant to dexamethasone-induced cell death. In striking contrast to the results with S49 Neo cells, the loss of cell viability in S49 Bcl-2 cells by serum depletion (1–2% FCS) was abrogated by dexamethasone treatment. This novel observation prompted us to more closely examine the type of cell death activated under our experimental conditions.
Analysis of the mode of cell death
The mode of cell death was assessed by flow cytometry in cells
cultured in different concentrations of FCS, using DNA content as an
index of apoptosis. The subdiploid peak reflected a decrease in
propidium iodide fluorescence, which corresponded to DNA fragmentation
during apoptosis (24
, 28
). Culture of cells
in decreasing concentrations of FCS resulted in an increase in
subdiploid DNA in both cell lines (Fig. 3
). Addition of dexamethasone to S49 Neo cells growing under all
serum conditions increased the amount of cells with subdiploid DNA
(Fig. 3)
. In contrast, treatment of dexamethasone in S49 Bcl-2 cells
cultured in 5% or 10% FCS-supplemented medium had only minimal
effects on the formation of subdiploid DNA while causing a marked
G1 arrest. A decrease in subdiploid peak formation after
dexamethasone treatment was observed in S49 Bcl-2 cells cultured in 1%
or 2% FCS-supplemented media, consistent with our observation that
glucocorticoid prevented death in Bcl-2-overexpressing cells under
conditions of serum withdrawal.
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To ascertain further if S49 Neo and S49 Bcl-2 cells were dying by necrosis and/or apoptosis, we also examined DNA degradation by agarose gel electrophoresis. (Fig. 4 A, B) Serum depletion induced internucleosomal DNA cleavage in S49 Neo. Dexamethasone also induced internucleosomal DNA cleavage in S49 Neo cells grown in 10% FCS-supplemented medium at all doses examined. In contrast and consistent with the flow cytometric and viability data described above, dexamethasone treatment did not induce a significant increase in internucleosomal DNA cleavage in S49 Bcl-2 cells cultured in 10% FCS-supplemented medium. Dexamethasone treatment also suppressed serum depletion-induced internucleosomal DNA cleavage in S49 Bcl-2 cells cultured in 1% FCS-supplemented medium.
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Caspase-3-like activity
Caspases have recently been implicated to be an important
component of the apoptotic machinery, including glucocorticoid-induced
apoptosis in lymphocytes (33)
. Bcl-2 prevents activation
of caspases by acting upstream of these enzymes (29)
;
therefore, we analyzed caspases-3-like activity under our experimental
conditions to elucidate potential interactions between the Bcl-2 and
glucocorticoid signaling pathways. Our data show that caspase-3-like
activity was induced by dexamethasone in S49 Neo cells cultured in 10%
FCS-supplemented medium (Fig. 5
). Induction of caspase-3-like activity by dexamethasone was not
observed in these cells grown in 1% FCS, although high levels of
caspase-3-like activity were induced by serum deprivation alone. In
contrast, caspase-3-like activity was not activated by dexamethasone in
Bcl-2-expressing cells cultured in 10% FCS where glucocorticoids
failed to induce apoptosis. Serum depletion, however, induced
caspase-3-like activity in Bcl-2 positive cells. The activity of
caspase-3-like enzyme induced by serum depletion in cells
overexpressing Bcl-2 was attenuated by all doses of dexamethasone,
implying that glucocorticoid signaling differs in the presence and
absence of Bcl-2.
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Effects of serum depletion and other apoptosis-inducing agents on
cell growth and cell death in lymphocytes
To elucidate possible mechanisms of the inhibitory effects of
glucocorticoids on serum depletion-induced apoptosis in lymphocytes, we
treated S49 Neo and S49 Bcl-2 cells with other apoptosis-inducing
agents under optimal growth conditions and after serum depletion.
Anisomycin (30)
, a protein synthesis inhibitor, inhibited
cell growth in both cell lines cultured in 10% FCS-supplemented
medium. There was no additional growth inhibitory effect by anisomycin
on either cell line cultured in 1% fetal serum-supplemented medium
(Table 1
A, B). S49 Neo cells underwent apoptosis in response to anisomycin
when cultured under high and low serum, whereas this compound did not
kill S49 Bcl-2 cells cultured in 10% serum-supplemented medium.
Consistent with the observations made with dexamethasone, anisomycin
inhibited the death of S49 Bcl-2 cells cultured in 1%
serum-supplemented medium.
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To further assess the potential involvement of inhibition of protein synthesis in protecting against serum depletion-induced apoptosis, we treated the cells with another protein synthesis inhibitor, cycloheximide (Table 1 A, B). Cell growth of both cell lines cultured in 10% serum-supplemented medium was inhibited by cycloheximide, whereas only S49 Neo cells cultured in 10% serum-supplemented medium were sensitive to cycloheximide-induced cell death. Cell death of S49 Bcl-2 cultured in 1% serum-supplemented medium was attenuated by cycloheximide treatment. Together, these data suggest that agents that inhibit protein synthesis can retard apoptosis in Bcl-2-expressing cells deprived of serum.
We next chose to determine whether agents that activate apoptosis by mechanisms other than inhibition of protein synthesis could also protect S49 Bcl-2 cells from serum depletion-induced apoptosis. Examination of both thapsigargin, an agent known to kill by elevation of intracellular Ca2+ (31) , and antibody against the Fas receptor (32 , 33 ), which also activates the caspase cascade, were completely ineffective (Table 1 A, B), arguing that protection of Bcl-2 signal cells against serum depletion-induced death is specific for apoptosis-inducing agents that act via repression of protein synthesis. Examination of another T cell line overexpressing Bcl-2, W.Hb12, resulted in similar findings to those observed for S49 Bcl-2 cells (unpublished observations), indicating that this protective effect of glucocorticoids may be a general property of T lymphocytes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast, the effects of serum deprivation and glucocorticoid treatment on induction of apoptosis in S49 Neo cells were additive. This result may be attributed to two possible causes. 1) The additive effect of apoptosis induced by serum depletion and glucocorticoids could be a summation from different cells with distinct sensitivities to various apoptotic stimuli or 2) the pathways of serum depletion- and glucocorticoid-induced apoptosis could be totally independent. Resistance of S49 Bcl-2 cells to dexamethasone-induced apoptosis has already been well-demonstrated; however, we have shown for the first time that apoptosis in S49 Bcl-2 cells can be induced by serum depletion. This result differs from myeloid cells expressing Bcl-2, which have been reported to be resistant to serum depletion-induced apoptosis (13) . Surprisingly, serum depletion-induced apoptosis in S49 Bcl-2 cells is partially attenuated by dexamethasone treatment. These effects are blocked by the receptor antagonist RU486, indicating that dexamethasone inhibits serum depletion-induced apoptosis in S49 Bcl-2 cells via a glucocorticoid receptor-dependent pathway (unpublished observations). Based on the results from cell growth and cell death in both cell lines, our data suggest that growth inhibition and apoptosis induction are two distinctly regulated processes.
Recently, members of the caspase family have been suggested to be key mediators in the apoptotic signaling pathway. The requirement for activation of caspase-3-like activity in apoptosis induced by glucocorticoids has also been demonstrated (34) . Bcl-2 is proposed to function upstream of caspase-3 (29) . Our data suggest that caspase-3-like activity is activated by either serum depletion or dexamethasone in S49 Neo cells, but only by serum depletion in S49 Bcl-2 cells. This caspase-3-like activity was decreased by dexamethasone when S49 Bcl-2 cells were cultured in serum-depleted medium. These results provide strong evidence that Bcl-2 can play an important role in regulating the proapoptotic actions of glucocorticoids.
Because glucocorticoids are well known to inhibit protein synthesis (23) , we also evaluated the effect of the protein synthesis inhibitors anisomycin and cycloheximide on apoptosis in lymphocytes. We demonstrated that, like glucocorticoids, anisomycin and cycloheximide protected Bcl-2-overexpressing cells cultured in serum-depleted media from apoptosis. Since the primary common effect of these agents is their ability to suppress protein synthesis, we speculate that dexamethasone, anisomycin, and cycloheximide may be inhibiting the synthesis of proteins required during the induction of apoptosis in Bcl-2-overexpressing cells. The net result would then be a conferred protection against apoptosis.
We have shown that Bcl-2-overexpressing lymphocytes cultured in media supplemented with 10% FCS are resistant to apoptosis induced by dexamethasone, anisomycin, and cycloheximide. However, we have also shown that these agents confer protection against apoptosis induced by serum depletion in these cells. If a cell becomes resistant to a particular apoptotic agent, is that agent necessarily protective against serum depletion-induced apoptosis? To answer this question, we treated the cells with anti-Fas and thapsigargin, since S49 Bcl-2 cells cultured in media supplemented with 10% fetal serum are resistant to anti-fas- but not to thapsigargin-induced apoptosis. Protection against serum depletion-induced apoptosis was found in neither anti-fas- nor thapsigargin-treated Bcl-2-overexpressing cells. These results suggest that the resistance to apoptosis and the conferred protective effect are not necessarily concomitant.
Our results also suggest that protection against serum depletion-induced apoptosis in S49 Bcl-2 cells may be related to protein synthesis inhibition. We next examined whether agents signaling through other mechanisms could also achieve this protection. Experiments in which we treated cells with anti-fas and thapsigargin could also address this question since these two agents do not inhibit protein synthesis, but, rather, activate caspase cascade signaling and increase intracellular calcium, respectively. As mentioned above, neither anti-fas nor thapsigargin protected S49 Bcl-2 cells from serum depletion-induced apoptosis. These data indicate that protein synthesis inhibition, and not an alteration of caspase cascade signaling or intracellular calcium concentration, is likely to be an important event associated with protection against serum depletion-induced apoptosis in S49 Bcl-2 cells.
It has been shown that the actions of glucocorticoids are cell type specific. Glucocorticoids have been demonstrated to induce apoptosis in some but not all T cells (35) . Therefore, we used another mouse T lymphoma cell line, Bcl-2-overexpressing WEHI cells (W.Hb12 cells), to examine cell specificity of the protective effects by glucocorticoids. The protective effects of glucocorticoids on appeared to be similar to those in S49 Bcl-2 cells (unpublished observations), suggesting that the protection may be a common phenomenon in T lymphocytes. Our data indicate that glucocorticoids can inhibit serum depletion-induced apoptosis in the presence of Bcl-2 protein, probably by attenuating the effect of apoptotic effectors or indirectly enhancing the function of apoptotic inhibitors. The ramifications of these findings suggest that glucocorticoids may be contraindicated in the treatment of lymphoid malignancies where Bcl-2 expression predominates.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 S.-T.J.H. is also a Ph.D. student of the
Department of Cell and Molecular Physiology at the University of North
Carolina at Chapel Hill, NC 27599, USA.
3 Abbreviations: AFC, 7-amino-4-trifluoromethylcoumarin;
DTT, DL-dithiothreitol; DMSO, dimethyl sulfoxide; FCS, fetal calf
serum; FMK, fluoromethylketone; FU, fluorescence units; HEPES,
N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid]; CHAPS,
3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; NP-40,
nonidet P-40; PBS, phosphate-buffered saline; TNF, tumor necrosis
factor; ICE, interleukin converting enzyme.
Received for publication June 22, 1998.
Revision received October 28, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 5% (
), 2% (
), or 1% (
) heat-inactivated FCS.
Viable cell number was measured by counting at a series of time points
(4, 8, 12, 24, 48 h). Cell cultures were started at 2 x
105 cells/ml. Data were reported as the mean ±
SEM; n=3. Paired t test: Significant
difference between 10% FCS and serum-depleted conditions (5%, 2%, or
1% FCS) is labeled with an asterisk (P<0.05). One-way
ANOVA: significantly different among 10%, 5%, 2%, and 1% FCS
(P<0.05).
), 0.0001
(
), 0.001
(
), 0.01
(
), 0.1 (
),
or 1 µM (
) and S49 Bcl-2 (