(Received for publication, March 20, 1997, and in revised form, April 30, 1997)
From the We report a novel mode of apoptosis induction
observed in human leukemic HL-60 cells. These cells spontaneously
underwent apoptosis in the course of proliferation when the cell
density became higher than 1 × 106/ml. This
occurred under ordinary in vitro culture conditions, with
or without fetal calf serum. Even the low density cells were committed
to undergo apoptosis if they were cultured under artificially concentrated conditions. Replacement of the culture supernatant of the
low density cells by that of the high density ones resulted in
apoptosis induction in the former cells. This apoptosis-inducing activity of the high density cell culture supernatant was completely eliminated by the action of trypsin but was fully restored following ultrafiltration by 3-kDa pore-sized membrane. A strong
apoptosis-inducing activity was recovered from the culture supernatant
of the high density HL-60 cells at a specific fraction in reverse-phase
column chromatography. Neither an interleukin- Apoptosis, or programmed cell death, is an important biological
process that is indispensable in normal development or differentiation, preservation of homeostasis, and liberation of hosts from viral attacks
or spread of malignant cells (1-3).
Apoptosis is observed under in vitro culture conditions as
well as in vivo. According to previous reports on its
induction in cultured cell lines, the modes of induction in
vitro fall into three categories. First, a wide variety of noxious
stimulations can bring on the condition. For example, addition of the
anticancer drugs actinomycin D (4) or etoposide (5) and cytotoxic
cytokines such as tumor necrosis factor Second, the absence of cytoprotective signals results in apoptosis in
particular cases. For example, rat pheochromocytoma-derived PC-12 cells
undergo apoptosis after the deprivation of nerve growth factor from the
culture medium (12), and the murine pro-B cell derived Ba/F3 cells do
so after the deprivation of interleukin-3 (13).
Finally, apoptosis is occasionally observed following the
differentiation of cells. For example, all-trans-retinoic
acid-treated human leukemic HL-60 cells are reported to undergo
apoptosis after having been differentiated to granulocytic
lineage (14).
In all these situations, however, certain positive or negative
environmental changes are required to induce apoptosis. It is not yet
clear whether exogenous forces which change the cellular environment
are always required, and to address this question, we investigated
whether the increase of cell density, which is an internal stress,
could possibly trigger apoptosis. We cultured human leukemic HL-60
cells under ordinary in vitro culture conditions and
examined apoptosis induction over a period of time.
In this paper, we show that these cells spontaneously undergo apoptosis
in the course of their proliferation without any external stress. We
also show evidence to prove that this spontaneous apoptosis is
triggered by an increase in cell density per se, which is a novel mode of induction. Finally, we show that the cell
density-dependent apoptosis is mediated by an unknown
soluble factor, present in the conditioned medium of these cells
themselves. Our work describes a novel mechanism of cell
auto-regulation: self-induced apoptosis in an inappropriately crowded
condition.
HL-60 cells were
maintained in RPMI 1640 medium (Life Technologies Inc.) supplemented
with 10% heat-inactivated fetal calf serum (JRH Bioscience, Lenexa,
KS). Cells were passaged every 2 days at an initial concentration of
6.5 × 104/ml. A bcl-2
overexpressing derivative line of HL-60 was the kind gift of Prof. S. Kitagawa and Dr. Y. Furukawa (15), and was maintained in 200 µg/ml
geneticin (Sigma). Recombinant human TGF- Small molecular weight chromosomal
DNA from the nucleus and also cytoplasm was prepared by Hirt's method
as originally described (16). Briefly, 3 × 106 cells
were lysed with 350 µl of lysis buffer containing 0.6% SDS and 0.1%
EDTA (pH 8.0), and after 10 min incubation at room temperature, 21 µl
of 5 M NaCl was added. This was mixed gently and incubated
at 4 °C for more than 8 h. It was then centrifuged at 15,000 rpm (MRX-150, Tomy Seiko Co., LTD., Tokyo, Japan) at 4 °C for 20 min, and the supernatant was further incubated with 1 mg/ml
heat-inactivated RNase A (Sigma) at 45 °C for 90 min and with 200 µg/ml proteinase K (Boehringer Mannheim, Mannheim, Germany) for
another 60 min. After phenol extraction, phenol/chloroform extraction,
and ethanol precipitation, one-third of the DNA sample was applied on
2% agarose gel, separated by horizontal electrophoresis, and
visualized by staining with ethidium bromide (500 ng/ml). Marker 4 (WAKO Chemicals Co., Ltd., Osaka, Japan) was used for the evaluation of
the molecular weights of the fragmented DNA.
In some experiments, cytoplasmic DNA was used to evaluate
apoptosis. 3 × 106 cells were lysed with 350 µl
of lysis buffer (5 mM Tris (pH 8.0), 0.5% Triton X-100, 10 mM EDTA), then centrifuged at 15,000 rpm at room
temperature for 20 min, and the supernatant was treated with RNase A
and proteinase K as described above.
The cells were washed with
phosphate-buffered saline and fixed on slide glasses using a cytospin
apparatus (Cytospin2, Shandon, Pittsburgh, PA). After being dried in
air and fixed with a mixture of acetone/methanol (3:1) for 5 min at
room temperature, the samples were incubated with 4 µg/ml H33342
(Calbiochem) for 10 min at room temperature. The chromatin structures
of the cells were examined by fluorescence light microscopy.
HL-60 cells were seeded
at an initial density of 1 × 105/ml in RPMI 1640 medium supplemented with 10% fetal calf serum. After 24 h of
incubation at 37 °C, the cells were washed and resuspended with
serum-free RPMI 1640 medium supplemented with 5 µg/ml bovine pancreas
insulin (Sigma) and 5 µg/ml human holo-transferrin (Sigma). Following
another 72 h incubation at 37 °C, the supernatant was collected
and stored at Type III trypsin
from bovine pancreas (Sigma) was suspended in 50 mM Tris at
pH 8.8 (i.e. 5% trypsin solution). We added 120 µl of
this solution to 3 ml of conditioned medium of HL-60 cells, incubated
it for 30 min at 37 °C, and then used it for the assay. For
inoculation of trypsin, this was ultrafiltrated by a 3-kDa pore-sized
membrane (Millipore Co., Bedford, MA) or boiled at 100 °C for 5 min.
Twenty-five ml of the conditioned
medium of HL-60 cells was diluted four times with 10 mM
Tris (pH 7.5). This diluted conditioned medium was then applied to a
20-ml column (Econopack column, Bio-Rad) packed with 5 ml of
CM-Sepharose (Sigma), which had previously been equilibrated with 10 mM Tris (pH 7.5). After the column was washed with 15 ml of
10 mM Tris (pH 7.5), the sample was eluted with 10 ml of
elution buffer (10 mM Tris (pH 7.5) with 150 mM NaCl). Seven ml of this elution was then applied to a reverse-phased column (Pro-RPC, Pharmacia Biotech, Uppsala, Sweden). The elution was
performed using a gradient mixture of water and acetonitrile, both of
which were supplemented with 0.1% trifluoroacetic acid. The elution
pattern was monitored by checking the absorbance value of the
ultraviolet wave (at a wavelength of 215 nm).
There appeared an increasing number of
dead cells among the in vitro cultured HL-60 cells with the
passage of time. In the initial experiment, we performed DNA
fragmentation assay and morphological examination to determine whether
they died by apoptosis.
Fig. 1A is the growth curve of HL-60 cells.
They exponentially grew as long as their density was less than 8 × 105/ml, and no sign of apoptosis was detected in the DNA
fragmentation assay (Fig. 1B, lanes 1 and 2) or
the morphological examination (Fig. 1C, upper). However,
apoptotic cells appeared when the cell density exceeded 1 × 106/ml (Fig. 1C, lower), in which
internucleosomal DNA fragmentation was actually detected (Fig.
1B, lanes 3 and 4). Similar results were obtained
when HL-60 cells were cultured in a serum-free condition and
supplemented with insulin and transferrin (data not shown). These
results indicate that HL-60 cells undergo apoptosis without known
apoptotic stimuli during in vitro culture.
To identify the mechanisms
of spontaneous apoptosis in HL-60 cells, we investigated whether the
increase of cell density per se could possibly be a trigger
for the apoptosis induction. The culture supernatant of the growing
phased cells (4 × 105/ml) was partially removed so
that the cells were in a 4 fold-concentrated condition (i.e.
1.6 × 106/ml). After 30 min incubation at 37 °C
they were diluted with fresh medium to the original concentration
(i.e. 4 × 105/ml), and after another
10 h incubation, DNA fragmentation assay was performed. For
controls, cells that were cultured without having been concentrated
were used.
As shown in Fig. 2, the cells that had been cultured in
a concentrated condition showed DNA fragmentation, whereas control cells did not show any fragmented DNA. This suggests that the apoptosis
commitment was made within the 30 min that HL-60 cells were cultured in
the artificially concentrated condition and that the "spontaneous
apoptosis" was strictly dependent on the cell density of the
culture.
To learn whether the cell
density-dependent apoptosis in HL-60 cells was mediated by
a soluble factor, the culture supernatant of low density cells (4 × 105/ml) was replaced by that of high density cells
(1.6 × 106/ml) in various proportions. After 10 h incubation at 37 °C, DNA fragmentation assay was performed. As
shown in Fig. 3A, addition of the high
density cell culture supernatant induced DNA fragmentation in the low
density cells in a dose-dependent manner. Similar results were obtained when this supernatant was prepared from cells maintained in a serum-free condition (data not shown). These results indicate that
the high density cells culture supernatant has an ability to induce
apoptosis in low density cells.
To determine whether the culture supernatant of the artificially
concentrated HL-60 cells also had an apoptosis inducing activity, the
growing phased cells (6 × 105/ml) were incubated in a
3-fold concentrated condition (1.8 × 106/ml) by
removing two-thirds of the culture supernatant. After incubation for 30 min or 6 h at 37 °C, the culture supernatants of these
artificially concentrated cells were collected, and their apoptosis
inducibility was assayed. The supernatant of the same growing phased
cells that had been cultured without concentration was used as control.
As shown in Fig. 3B, addition of the artificially concentrated cells' culture supernatant caused apoptosis in the growing phased cells, suggesting that the artificially concentrated HL-60 cells release an inducer of apoptosis into the medium within a
short period of cultivation in response to the high cell
concentration.
To characterize
the soluble apoptosis-inducing activity in the conditioned medium of
HL-60 cells, we studied the effect of trypsin treatment of the high
density cell culture supernatant on the induction. The conditioned
medium, which was prepared from HL-60 cells cultured in a serum-free
condition, was treated with trypsin or buffer only, and the
apoptosis-inducing activity was assayed. As shown in Fig.
4, the trypsin-treated culture supernatant did not
induce apoptosis at all in the exponentially growing phased cells. This
finding indicates that the cell density-dependent apoptosis
in HL-60 cells is mediated by a soluble factor that is sensitive to
trypsin treatment. In examining the chemical or mechanical stability of
this apoptosis-inducing activity, we found that it was completely
resistant to mild (56 °C, 30 min) and severe heat treatment
(100 °C, 5 min) and also to transient alkaline (pH 11) or acid (pH
3.5) treatment (data not shown).
To further confirm the existence of certain apoptosis-inducing
molecules in the culture supernatant, we partially purified this
factor. The HL-60 cell conditioned medium was first semi-purified by
anion exchange column chromatography. Twenty-five ml of the medium was
diluted four times with salt-free buffer and applied to CM-Sepharose
column, and stepwise elution was performed using buffer containing 150 mM, 500 mM, or 1000 mM NaCl.
Because we found that the apoptosis-inducing activity was concentrated
in the 150 mM NaCl-eluted fraction (data not shown), we
applied this fraction to reverse-phased column chromatography, and
gradient elution was performed using acetonitrile/water solution (Fig. 5A). Each eluted fraction, suspended in 1 ml
of RPMI 1640 medium, was added to the exponentially growing HL-60
cells, and apoptosis-inducing activity was evaluated by DNA
fragmentation assay as before. As shown in Fig. 5B,
relatively strong activities were detected in the fifth and also the
sixth fraction. For a more quantitative evaluation, the number of dead
cells was counted. As shown in Fig. 5C, the strongest
activity was detected in the sixth fraction. These results indicate
that the cell density-dependent apoptosis in HL-60 cells is
mediated by a specific soluble factor.
To determine the rough molecular weight of this factor, the conditioned
medium of HL-60 cells was ultrafiltrated through 3-, 5-, 10-, 30-, or
100-kDa pore-sized membrane, and the apoptosis-inducing activity of
each flow-through fraction was evaluated by DNA fragmentation assay. As
shown in Fig. 5D, the material ultrafiltrated through 3-kDa
pore-sized membrane had as strong an activity as the crude conditioned
medium. Similar results were obtained when membranes of other pore
sizes were used (data not shown). This finding suggests that the
mediator of the cell density-dependent apoptosis is a relatively small molecule less than 3 kDa in molecular mass.
Investigation of the effects of cytotoxic cytokines or
growth inhibitory factors on the cell density-dependent
apoptosis in HL-60 cells showed that TNF-
The anti-apoptotic effect of TGF- To explore whether the
overexpression of Bcl-2 protein could inhibit the induction of the cell
density-dependent apoptosis in HL-60 cells, two derivative
lines of these cells, in which the expression vector of
bcl-2 gene or neomycin-resistant gene was stably transfected
(15), were cultured, and the DNA fragmentation assay was performed in
accordance with the time course. Overexpression of Bcl-2 protein did
not affect the growth rate of the cells (Fig. 7A); however, it markedly attenuated the
induction of cell density-dependent apoptosis (Fig.
7B).
The resistance to cell density-dependent apoptosis in Bcl-2
overexpressing HL-60 cells was due either to the decreased production of apoptosis-inducing factor or the decreased sensitivity to this factor. To address this question, conditioned media of Bcl-2
overexpressing and the control neomycin-resistant HL-60 cells were
added to the exponentially growing parental HL-60 cells, and DNA
fragmentation assays were performed as before. As shown in Fig.
7C, the conditioned media of both cell lines showed similar
apoptosis-inducing activities, suggesting that the overexpression of
Bcl-2 protein did not inhibit production of the inducer but blocked the
intracellular signaling events that lead to the induction.
Finally, to determine the possible involvement of ICE-
or CPP-32-dependent pathways in cell
density-dependent apoptosis, HL-60 cells were cultured with
or without the inhibitor against ICE or CPP-32, and DNA fragmentation
assay was performed at almost the confluence phase. Neither ICE nor
CPP-32 inhibitor rescued but, rather, both slightly enhanced the
induction of apoptosis (Fig. 8A, lanes
2 and 3). Under the same conditions, both inhibitors potently suppressed the TNF-
We demonstrated that human myeloblastic HL-60 cells underwent
apoptosis strictly dependent on the cell density during in
vitro culture with or without fetal calf serum. The cell
density-dependent apoptosis was, at least in part, mediated
by a soluble factor, and the autocrine or paracrine mediator was found
to be a low molecular weight peptide-containing molecule. In regard to
the intracellular mechanisms of this novel mode of apoptosis,
ICE-related pathways, ICE by itself and its downstream molecule of
CPP-32, are not likely to be involved, but the Bcl-2-sensitive
component would contribute to the intracellular signaling of cell
density-dependent apoptosis. Interestingly, TGF- As shown, this apoptosis-inducing factor seems to be a small peptide
whose molecular mass is less than 3 kDa. Our preliminary observation
shows that the factor is even smaller than 1 kDa, which indicates that
it may be composed of only 5 to 10 amino acids. There are various kinds
of physiologically active oligopeptides, including hormones and
neurotransmitters. Among these substances, a pentapeptide called
hematoregulatory peptide (HP), which was isolated from the conditioned
media of mature granulocytes as an inhibitor against the formation of
myelopoietic colonies, was reported to inhibit the growth of HL-60
cells (reviewed by Paukovits et al. (17)). We do not know
whether HP can also induce apoptosis in HL-60 cells. However, it is not
likely that this peptide would be the factor we are trying to isolate,
because HP is reported to be completely resistant to the action of
trypsin.
Are any cytotoxic cytokines involved in the induction of the cell
density-dependent apoptosis in HL-60 cells? It is reported that TNF- TGF- However, we observed a completely unexpected result: TGF- The mechanism of this inhibition by TGF- It has also been reported that TGF- We showed that the overexpression of Bcl-2 protein strongly inhibited
the induction of cell density-dependent apoptosis in HL-60
cells. We also showed that the conditioned medium of Bcl-2 overexpressing HL-60 cells had as well an activity to induce apoptosis in parental cells, indicating that Bcl-2 protein did not inhibit the
release of the apoptosis-inducing factor but, rather, antagonized the
intracellular signaling pathway for apoptosis induction. It is also of
interest whether other Bcl-2 family proteins, including Bcl-x and Bax,
can inhibit HL-60 cells spontaneous apoptosis.
Whether cell density-dependent apoptosis is a phenomenon
specific to HL-60 cells or is more generalized is an interesting point.
We also investigated the existence of cell
density-dependent apoptosis using two other human leukemic
lines, U937 cells and MO7e cells, and we observed that MO7e cells
underwent apoptosis in the course of proliferation when the cell
density rose above 7 × 105/ml (data not shown).
Interestingly, cell density-dependent apoptosis in these
cells was not affected by TGF- Cell density-dependent apoptosis is physiologically of
great importance not only in vitro but also in
vivo, since it may contribute to the homeostasis of blood cell
production via a negative feedback regulator of cell proliferation.
Mechanistic study of cell density-dependent apoptosis may
also lead to the development of a novel therapy for hematological
malignancies. Further studies are required to prevent the uncontrolled
proliferation of tumor cells by the application of cell
density-dependent apoptosis.
We thank Prof. Kitagawa at Osaka City
University and Dr. Y. Furukawa at Jichii Medical School for the kind
gift of Bcl-2-transfected HL-60 cells and E. Okuma for technical
support.
Department of Hematology,
The Third Department of Internal
Medicine,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
converting enzyme
inhibitor nor CPP-32 inhibitor blocked the induction of cell
density-dependent apoptosis in HL-60 cells, although
overexpression of Bcl-2 protein markedly attenuated the induction of
this mode. Surprisingly, transforming growth factor-
1 and activin A
did not induce but, rather, inhibited the induction of cell
density-dependent apoptosis. These data suggest that HL-60
cells release an unknown low molecular weight peptide-containing factor
in response to an increase in cell density to induce apoptosis in an
autocrine manner and that the interleukin-
converting
enzyme-independent intracellular machinery for this mode of apoptosis
is strongly affected by signaling events through the transforming
growth factor-
1 receptor and by the action of Bcl-2 oncoprotein.
(TNF-
)1 (6) or transforming growth
factor
1 (TGF-
1) (7) to the culture media of hematopoietic cells
results in apoptosis. The high osmotic pressure of the culture medium
also induces apoptosis in epithelial cell lines (8). In addition, heat
shock stimulation (9) and ultraviolet (10) or gamma ray irradiation
(11) induce apoptosis in some cases.
Cells, Cytokines, and Inhibitors
1 and activin A were
generously provided by Kirin Brewery Co., Ltd. (Tokyo, Japan) and
Ajinomoto Company Inc. (Kawasaki, Japan), respectively. Recombinant
human TNF-
was purchased from Pepro Tech EC, Ltd. (London, UK). The
interleukin-1
converting enzyme (ICE) inhibitor II (Bachem
Feinchemikalien AG, Bubendorf, Switzerland) and CPP-32 inhibitor
(Peptide Institute Inc., Osaka, Japan) were dissolved in
Me2SO and kept at
20 °C until use.
80 °C until use.
HL-60 Cells Spontaneously Underwent Apoptosis during the Course of
Proliferation in Vitro
Fig. 1.
Growth and apoptosis induction in HL-60
cells. A, growth curve. HL-60 cells were seeded at an
initial concentration of 6.5 × 104/ml and incubated
at 37 °C for 25 h, at which time the cell density reached
3.5 × 105/ml (time = 0). After further
incubation for indicated periods, the cell numbers were counted.
B, DNA fragmentation assay. Small molecular weight DNA was
extracted from the nucleus and cytoplasm of the cells when the cell
density of HL-60 cells reached the indicated number. Timings of cell
harvest are shown by arrows in A. The existence
of the internucleosomal DNA fragmentation was examined by agarose gel
electrophoresis (2% gel). C, morphology of the cells. Two
cell cultures at the density of 4.5 × 105/ml
(upper) and 1.2 × 106/ml
(lower) were fixed on slide glasses and stained with Hoechst 33342 (4 µg/ml). The nuclear morphology of the cells was examined by
fluorescence light microscopy (100 ×).
[View Larger Version of this Image (45K GIF file)]
Fig. 2.
Apoptosis commitment by an artificial
concentration of HL-60 cells. The exponentially growing cell
culture (3.5 × 105/ml) was centrifuged, and the
culture supernatant was partially removed. Cells were then resuspended
with the residual supernatant to a concentration of 2 × 106/ml. After 30 min incubation at 37 °C, fresh medium
was added to return them to the original density (3.5 × 105/ml). After a further 10-h incubation, DNA fragmentation
assay was performed as in Fig. 1. Control 1 represents the
chromosomal DNA of HL-60 cells cultured without centrifugation or
concentration, and Control 2 represents those with
centrifugation but without concentration.
[View Larger Version of this Image (36K GIF file)]
Fig. 3.
Conditioned medium of HL-60 cells has the
ability to induce apoptosis. A, effect of the conditioned
medium of naturally growing HL-60 cells. 10-ml culture of exponentially
growing phased HL-60 cells (4 × 105/ml) was
centrifuged, and the culture supernatant was partially replaced by that
of the high density cells (1.6 × 106/ml) at the
proportions indicated. After being resuspended with this mixture of
supernatant, cells were incubated at 37 °C for 10 h, and DNA
fragmentation assay was performed as in Fig. 1. B, effect of
the conditioned medium of artificially concentrated HL-60 cells. The
exponentially growing phased HL-60 cells (4 × 105/ml)
were centrifuged, and the culture supernatant was partially removed and
resuspended so that cells were concentrated to 1.6 × 106/ml. After 30 min (lane 2) or 6 h
incubation (lane 3) at 37 °C, the culture supernatants
were collected and their apoptosis-inducing activity was examined by
adding them to the exponentially growing phased cells as in Fig. 2. As
a control (lane 1), the exponentially growing responder
cells were centrifuged and resuspended without replacing the
supernatant.
[View Larger Version of this Image (29K GIF file)]
Fig. 4.
Trypsin treatment completely eliminated the
apoptosis inducibility of HL-60 cells' conditioned medium. The
exponentially growing phased cells (4 × 105/ml) were
centrifuged and resuspended in their own supernatant (lane
1), or half of their culture supernatant was replaced by that of
the cells that had grown to confluence in serum-free RPMI 1640 medium
supplemented with insulin and transferrin (lane 2). After
10 h incubation at 37 °C, DNA fragmentation assay was performed as in Fig. 1. In lane 3, the confluent phased cells'
culture supernatant had previously been treated with trypsin or with
buffer only (lane 4), before adding it to the exponentially
growing responder cells (see "Materials and Methods").
[View Larger Version of this Image (37K GIF file)]
Fig. 5.
Partial purification of apoptosis-inducing
activity from the conditioned medium of HL-60 cells. A,
elution pattern in hydrophobic column chromatography. 25 ml of
conditioned medium of HL-60 cells, which had been previously
semi-purified by CM-Sepharose column chromatography (see "Materials
and Methods"), was applied to a hydrophobic column (Pro-RPC,
Pharmacia) and eluted by a mixture of water and acetonitrile. The
elution pattern was monitored by the absorbance value at 215 nm. Before
the assay, each fraction was completely dried to remove acetonitrile
and suspended with 1 ml of RPMI 1640 medium. B,
apoptosis-inducing activity of each fraction as determined by DNA
fragmentation assay. 4-ml cultures of the exponentially growing phased
HL-60 cells (6 × 105/ml) were centrifuged, and 1 ml
of their supernatant was removed, to which each eluted fraction in
A suspended in 1 ml of RPMI 1640 medium was added. After
being suspended with the mixture of their residual supernatant and the
eluted fraction, the exponentially growing responder cells were
incubated at 37 °C for 10 h, and DNA fragmentation assay was
performed as in Fig. 1. In lane 8, 1 ml of the crude
conditioned medium (starting material) was used for the assay.
C, apoptosis-inducing activity of each fraction as
determined by morphological examination. The number of apoptotic featured cells was counted before the extraction of the chromosomal DNA
in B, and apoptosis-inducing activity of each fraction was evaluated by morphology of responder cells. D, evaluation of
the molecular weight. The conditioned medium was ultrafiltrated through 3-kDa pore-sized membrane, and the apoptosis-inducing activity of the
flow-through fraction was assayed as in B (lane
3). In lane 2, crude conditioned medium was used as a
positive control.
[View Larger Version of this Image (39K GIF file)]
1 and Activin A Inhibited the
Induction of the Cell Density-dependent Apoptosis in HL-60
Cells
, which is known to
induce apoptosis in human leukemic U937 cells (6), did not induce
apoptosis by itself and neither enhanced nor suppressed the cell
density-dependent apoptosis in HL-60 cells (data not
shown). We also found that TGF-
1 and activin A did not induce but,
rather, strongly inhibited cell density-dependent apoptosis
in HL-60 cells. As shown in Fig. 6A, TGF-
1
did not exert any effect on the proliferation of the exponential growth
of low density HL-60 cells. However, in its presence these cells showed
apparent growth advantage as compared with those without TGF-
1 after
cell density of the culture exceeded 1 × 106/ml, a
critical point of cell density-dependent apoptosis of HL-60 cells; these cells with TGF-
1 finally reached the viable cell concentration of 2~3 × 106/ml, which was never
observed under an ordinary culture condition. These findings strongly
suggest the inhibition of cell density-dependent apoptosis
by TGF-
1, and in fact, potent inhibition of DNA ladder formation by
this factor was clearly demonstrated in HL-60 cells at high cell
density (Fig. 6B). Similar results were obtained when HL-60
cells were cultured with activin A (Fig. 6, C and
D), suggesting that this interesting and unexpected
phenomenon is a common and specific action of a member of the TGF-
family of cytokines. These findings for TGF-
1 and activin A were
further confirmed by morphological examination of the cells (Fig.
6E, data for activin A are not shown).
Fig. 6.
Transforming growth factor 1 (TGF-
1)
and activin A blocked the induction of cell
density-dependent apoptosis in HL-60 cells. A
and C, growth curves. HL-60 cells were seeded at an initial
concentration of 1 × 105/ml with or without cytokines
(continuous line, buffer only; broken line in
A, 10 ng/ml TGF-
1; broken line in
C, 20 ng/ml of activin A). After incubation at
37 °C for the indicated periods, the cell numbers were counted.
B and D, TGF-
1 and activin A inhibit cell density-dependent apoptosis. Small molecular weight DNA was
extracted from the cell cultures indicated by arrows in
A and C as in Fig. 1 and separated by agarose gel
electrophoresis. E, morphological examination of
TGF-
1-treated cells. Control cells (above) or TGF-
1-treated cells (below) were collected at the
indicated time (arrow in A), fixed, and stained
with H33342. F, apoptosis inducibility of conditioned medium
(C.M.) was not reduced by TGF-
1. HL-60 cells
were cultured with or without TGF-
1 to the confluence phase (arrows in A), and the culture supernatants were
collected and ultrafiltrated through 3-kDa pore-sized membrane. 5-ml
culture of the exponentially growing phased cells (6 × 105/ml) was centrifuged and resuspended (lane
1), or 3 ml of the supernatant was removed and 3 ml of
TGF-
1(
)- (lane 2) or TGF-
1(+) (lane
3)-conditioned medium was added. After 12 h incubation at 37 °C, DNA fragmentation assay was performed as in Fig. 1.
G, TGF-
1 inhibits the conditioned medium-induced
apoptosis in HL-60 cells. 4-ml culture of the exponentially growing
phased cells (4 × 105/ml) was centrifuged and
resuspended (lane 1), or 1 ml of the supernatant was removed
and 1 ml of the conditioned medium added (lane 2). In
lane 3, 1 ml of conditioned medium was added together with
10 ng/ml TGF-
1. After 10 h incubation at 37 °C, DNA
fragmentation assay was performed as in Fig. 1.
[View Larger Version of this Image (58K GIF file)]
1 may be due either to HL-60
cells' decreased production of apoptosis-inducing factor or their
acquisition of resistance to it. To address this question, HL-60 cells
were cultured with or without TGF-
1, and the culture supernatant was
prepared, ultrafiltrated through 3-kDa pore-sized membrane to deplete
any residual TGF-
1, and added to the exponentially growing cells to
evaluate the apoptosis-inducing activity. As shown in Fig.
6F, coexistence of TGF-
1 did not reduce the apoptosis inducibility of the conditioned medium, suggesting that
TGF-
1-treated cells still release apoptosis inducer into the culture
medium without being compelled to undergo apoptosis themselves. We also examined the effect of TGF-
1 on the apoptosis induced by exogenously added soluble inducer. As shown in Fig. 6G, TGF-
1 almost
completely abrogated DNA ladder formation in HL-60 cells cultured with
conditioned media of high cell density culture. These findings clearly
indicate that TGF-
1 inhibits cell density-dependent
apoptosis not by simple reduction of the release of soluble apoptosis
inducer but, rather, by interaction of its intracellular signaling
pathways with those activated by high cell density and/or soluble
inducer.
Fig. 7.
Overexpression of Bcl-2 protein inhibits the
progression of the cell density-dependent apoptosis in
HL-60 cells. A, growth curves. The Bcl-2 overexpressing
derivative of HL-60 cells (Bcl-2) or neomycin-resistant control cells
(neor) was seeded at an initial concentration of 2 × 105/ml, and the cell numbers were counted over time
(continuous line, neor; broken line,
Bcl-2). B, DNA fragmentation assay. Small molecular weight
DNA was extracted from neor (left) or Bcl-2
(right) transformant line at indicated time points in
A (arrows), when the cell density reached the
indicated numbers in B, and separated by agarose gel
electrophoresis (2% gel). C, effects of conditioned medium
(C.M.) of Bcl-2 overexpressing cells. Bcl-2
overexpressing HL-60 derivative cells or neor control cells
were grown almost to confluence, and their culture supernatant was
added to the exponentially growing phased parental HL-60 cells. After
16 h incubation, only the cytoplasmic DNA was extracted and
separated by agarose gel electrophoresis.
[View Larger Version of this Image (23K GIF file)]
Converting Enzyme (ICE) nor CPP-32
Inhibitor Rescued the Cell Density-dependent
Apoptosis
-mediated apoptosis in U937 cells (Fig. 8B). Thus, the cell density-dependent
apoptosis in HL-60 cells may be mediated by ICE/CPP-32 independent
pathways.
Fig. 8.
Neither ICE nor CPP-32 inhibitor rescued the
cell density-dependent apoptosis. A, effects of inhibitors
on cell density-dependent apoptosis. 46 h after from the start of
cell culture, when the cell density of HL-60 cells had reached 6 × 105/ml, 0.2% Me2SO alone (lane 1, D), 200 µM ICE inhibitor (lane 2, I) or
200 µM CPP-32 inhibitor (lane 3, C) was added.
After incubation for another 45 h, cytoplasmic DNA was prepared.
B, effects of inhibitors on TNF--induced apoptosis.
46 h after the start of cell culture, when the cell density of
U937 cells reached 4.8 × 105/ml, 0.2%
Me2SO alone (lanes 1 and 2,
D), 200 µM ICE inhibitor (lane 2, I) or 200 µM CPP-32 inhibitor (lane 3, C)
was added, followed by the delayed addition of TNF-
(100 ng/ml) 30 min later (lanes 2-4). After incubation for another 15 h, cytoplasmic DNA was extracted.
[View Larger Version of this Image (36K GIF file)]
family
cytokines potently inhibited this novel mode of apoptosis in HL-60
cells.
induced apoptosis in these cells. However, we noted that a
high concentration of TNF-
(100 ng/ml) induced only a weak
smear-formed DNA breakdown with minimal ladder formation even after 2 days of incubation (data not shown); this would suggest that TNF
receptor (TNFR)-mediated pathways may not be involved in the induction
of cell density-dependent apoptosis in HL-60 cells. This
speculation is further supported by our observation that this apoptosis
was effectively blocked by the overexpression of Bcl-2 protein, because
it is reported that Bcl-2-overexpressing HL-60 cells are sensitive to
TNFR-mediated apoptosis (18). Similarly, the Fas-mediated pathways
would also not be involved, since anti-Fas antibody did not cause any
DNA breakdown at all (data not shown). It is known that TNFR- and
Fas-mediated apoptosis are mediated by ICE-dependent
pathway. Our observation that neither an ICE nor CPP-32 inhibitor
blocked the cell density-dependent apoptosis in HL-60 cells
confirms our speculation.
1 is also known as an apoptosis-inducing cytokine, and it has
been reported to induce apoptosis in hematopoietic cells (7, 19, 20),
fibroblasts (21), and epidermal cells (22-24). However, it is not yet
understood how the signals for this induction are transmitted through
the TGF
receptor (TGF
R). Recently, a novel mitogen-activated
kinase kinase kinase and mitogen-activated kinase kinase were cloned
that mediate the signals from TGF
R (25, 26). Because
mitogen-activated kinase kinase kinase-mediated signaling pathways are
reported to be involved in the induction of apoptosis in rat PC-12
cells (27) and also in mink Mv1Lu cells (28), it seems likely that
TGF-
1 induces apoptosis by activating these pathways.
1 strongly
inhibited the induction of the cell density-dependent apoptosis in HL-60 cells. There is only one report mentioning the
anti-apoptotic effect of TGF-
1, where it is shown that the apoptosis
of peripheral T cells in T cell receptor-transgenic mice (29) was
inhibited by the coexistence of TGF-
and interleukin-2. But even in
this case, TGF-
added alone without interleukin-2 induced apoptosis
(29). Our result is thus the first that clearly illustrates the sole
contribution of TGF-
1 to apoptosis inhibition. Furthermore, this
anti-apoptotic effect of TGF-
1 seems to be specific for the cell
density-dependent apoptosis in HL-60 cells, because it did
not inhibit but, instead, enhanced the actinomycin D-induced apoptosis
in these cells (data not shown). In examining the effect of activin A,
a member of the TGF-
superfamily, we found that it also inhibited
the induction of the cell density-dependent apoptosis in
HL-60 cells.
1 in HL-60 cells is a
matter of great interest. Decreased production of autocrine apoptosis-inducer by TGF-
1 was ruled out (Fig. 6F), and
its antagonistic effect on the apoptosis inducer was confirmed (Fig.
6G). These results suggest two possibilities for the
mechanism of the inhibitory effect on cell
density-dependent apoptosis. One is the rapid interaction of intracellular signaling events triggered by TGF-
1 with those by
the apoptosis inducer. The other is the more delayed effect of
TGF-
1, probably via synthesis of certain anti-apoptotic proteins. According to our preliminary studies, TGF-
1 had no effect on the
protein expression of well-known anti-apoptotic genes, including bcl-2, bcl-xL, and hcl-1. Further
investigations from the standing point of signaling transduction and
also new gene induction are required.
1 was able to inhibit cell
proliferation (30); this seems to be achieved by the arrest of the cell
cycle progression (31). In fact, we observed inhibition of
proliferation (decrease in cell cycle progression speed) in human
leukemic MO7e cells by TGF-
1 (data not shown). We demonstrated earlier, however, that TGF-
1 had no effect on proliferation speed (progression of cell cycle) in HL-60 cells despite its potent inhibitory effect on apoptosis. These findings together indicate that
TGF-
1 affects the cell cycle progression or apoptosis induction according to the cell types and also suggest that the signaling pathways for cell cycle arrest and those for apoptosis inhibition are
independently regulated.
1 (data not shown). U937 cells, on the
other hand, did not undergo apoptosis in the course of normal
proliferation, but did when cultured in an artificially concentrated
condition where the cell density was higher than 3 × 106/ml. Furthermore, this
concentration-dependent apoptosis induction in U937 cells
was inhibited by TGF-
1 (preliminary observation). Thus, cell
density-dependent apoptosis seems to be a common phenomenon in human hematopoietic cells, although there are some differences in
the mode of its induction among cell lines and its sensitivity to the
actions of TGF-
1.
*
This work was supported by a Grant-in-Aid from the Ministry
of Education, Science and Culture, 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: Dept. of Hematology,
Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162, Japan. Tel.: 81-3-3202-7181 (ext.
2807); Fax: 81-3-3207-1038.
1
The abbreviations used are: TNF-, tumor
necrosis factor
; TGF-
1, transforming growth factor
1; HP,
hematoregulatory peptides; ICE, interleukin-1
converting enzyme; R,
receptor.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.