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Tumor Biology |
Department of Retroviral Regulation, Medical Research Division, Tokyo Medical and Dental University, Tokyo 113-8519 [H. S., Y. I., N. O.], and Department of Immunology, Juntendo University School of Medicine, Tokyo 113-8421 [H. Y.], Japan
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Fas-mediated signal transduction pathways have been extensively studied over the last several years. The Fas-mediated signaling activates the caspase cascade that ultimately results in intracellular proteolysis and death (reviewed in Refs. 1 and 9 ). An alternate signaling pathway downstream of Fas involves the MAPK3 family members. Three distinct but related kinase cascades have been identified: the ERKs, the JNKs/stress-responsive MAP kinases, and the p38 MAPK. ERK is primarily activated by mitogens and growth factors, whereas JNK and p38 are stimulated by various stresses. Some previous studies showed that Fas triggered activation of MAPK, including JNK, p38, and ERK (10, 11, 12, 13, 14, 15, 16) . In Jurkat T cells, Fas cross-linking triggered JNK and p38 activation in a caspase-dependent manner (10, 11, 12 , 16) . During Fas-mediated apoptosis, caspases cleaved and activated PAK2 (15) and MAPK/ERK kinase kinase 1 (12) , two kinases that can activate the JNK pathway. Fas cross-linking was also shown to activate ERK in SHEP cells (14) , presumably via activation of the Ras pathway. Moreover, the additional connection of the Fas receptor to the stress-activated kinase pathway has recently been elucidated that upon Fas ligation, Daxx, a novel Fas-binding protein, interacts with and activates a MAP kinase kinase kinase termed ASK1, leading to the activation of the JNK and p38 MAPK pathways (17) . Collectively, Fas can activate MAPK pathways by divergent mechanisms, some of which are caspase-dependent or -independent. However, the biological role of MAPK activation downstream of Fas is presently controversial and needs further clarification.
Ligation of Fas with FasL dose not solely transmit a death signal but
instead may induce cell proliferation. In naïve T lymphocytes,
for example, Fas provided a costimulatory signal for proliferation yet
also induced apoptosis in repeatedly stimulated T lymphocytes
(18)
. Fas ligation has also been shown to enhance the
growth of some tumor cell lines (19)
. However, the
signaling mechanisms for proliferative effects of Fas ligation have not
been clarified yet. Human gliomas are the most common brain tumors that
possess unique characters with respect to cytokine sensitivities. These
tumors express tumor necrosis factor 
and its receptors and
furthermore, proliferate in response to this cytokine (20
, 21)
. It has been reported and we now show that some glioma cells
express both Fas (22)
and FasL (8)
, but the
pathophysiological role of this endogenously expressed Fas/FasL
interaction is not known. Although expressing substantial levels of
Fas, glioma cells are generally resistant to cell death induced by
anti-Fas treatment (22)
. Here, we show that endogenous
Fas/FasL interaction provides a growth, rather than cell death, signal
in glioma cells. Further, using Fas-transfected gliomas, we show
evidence that Fas definitely transmits a cell cycle progression signal
that is closely linked to ERK activation. We propose an additional role
of Fas-FasL in tumorigenesis, that is, some tumors may make use of this
system not only for evading immune surveillance but also for its own
growth.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies and Reagents.
Agonistic antihuman Fas mAb (clone CH-11; IgM) was purchased from MBL
(Nagoya, Japan). Mouse mAb to human FasL (NOK-2; IgG2a) was prepared as
described (23)
. Control mouse IgG2a for NOK-2 and control
mouse IgM for CH-11 were purchased from PharMingen (San Diego, CA). PI
was from Sigma (St. Louis, MO). 7AAD was from Molecular Probes (Eugene,
OR), and PY was from Wako Pure Chemical (Osaka, Japan). Rh123 and the
MEK inhibitor, PD98059, were from Calbiochem (La Jolla, CA). A
broad-spectrum caspase inhibitor, zD, was from Peptide Institute Inc.
(Osaka, Japan).
Estimation of Viability and Cell Death.
A modification of MTT assay was applied for estimating cellular
viability using a commercially available kit [Cell Counting Kit-8,
which contains dye WST-8
(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,
monosodium salt (Wako Pure Chemical)]. In brief, cells were seeded at
2 x 104 cells/well in 96-well
plates. The next day, anti-Fas (CH-11) was added at the given
concentrations, and the WST-8 was added the last 2 h before the
end of culture. The cell-bound dye was measured by optical absorption
at 450 nm using a microplate reader. Cell viability after CH-11
treatment was calculated as follows: % viability = (A450/640 CH-11-treated
cells/A450/640 untreated
cells) x 100. Rh123 and PI were used for the
determination of mitochondrial membrane potential (
m) and loss of
plasma membrane integrity, respectively, as described
(24)
. In brief, cells were incubated with Rh123 at 5
µg/ml for 30 min at 37°C, washed with PBS, and resuspended in PBS
containing PI at 2 µg/ml. Rh123 and PI staining were analyzed on a
flow cytometry (FACS Calibur, Becton Dickinson, San Jose, CA) using
CELLQuest software (Becton Dickinson). Trypan blue exclusion was also
used for counting of live cells under microscopy.
Transfection.
Preparation and transfection of retrovirus were performed as previously
described (25)
. Briefly, the full-length human Fas cDNA
(provided by Dr. S. Nagata, Osaka University, Japan) was digested by
XhoI and inserted into a MuLV retroviral vector PLXSN that
contains neomycin-resistant gene as a selection marker. This construct
was then transfected into the amphotropic packaging cell line PA317
with a commercially available kit (SuperFect, QIAGEN, Hilden, Germany).
Supernatant from the PA317 was used to infect three glioma cell lines,
A-172, T98G, and YKG-1, respectively in the presence of G418 (500
µg/ml). Two weeks after infection, each G418-resistant colony was
picked up and expanded as a clonal population. Vector controls from
each cell line that contain PLXN backbone were prepared in the same
way.
Immunofluorescent Staining and Flow Cytometry.
For study of cell surface expression of Fas and FasL, cells were
stained with FITC-conjugated anti-Fas mAb (clone UB2; MBL) and
phycoerythrin-conjugated anti-FasL mAb (NOK-2; Ref.
23
). FITC- or PE-conjugated isotype control IgGs were
obtained from PharMingen. The stained cells were analyzed on a flow
cytometry.
Cell-mediated Cytotoxic Assay.
A human Fas-transfected murine lymphoma cell line (WR19L/Fas) was used
as the target cells (23)
. Cytotoxic activities of glioma
cells were estimated by measuring target cell DNA fragmentation using
the JAM test as described (5)
. In brief, the
effector glioma cells were seeded onto a 24-well culture dish and
[3H] TdR (Amersham Pharmacia Biotech,
Buckinghamshire, England) -labeled WR19L/Fas target cells were added at
the given E:T ratios. After coculture for 16 h, intact DNA
was collected by precipitation with trichloroacetic acid and was
washed through the membrane filter (Millipore Corporation, Bedford,
MA). Specific killing was calculated as follows: [(cpm without
effector - cpm with effector)/cpm without
effector] x 100.
Cell Proliferation Assay.
Cells were seeded in a 48 well-dish at a concentration 1 x 105. After 16-h incubation, medium was
removed, and the cells were cultured with fresh medium containing 1
µCi/ml of [3H]TdR and 100 ng/ml CH-11 for
24 h as described (20)
.
Quantitation of Cellular DNA and RNA Contents.
Cell cycle analysis was performed using nucleic acid dyes, 7AAD, and
PY, which enable quantitation of the content of DNA and RNA separately
as described (26)
. In brief, trypsin-detached cells were
suspended in nucleic acid staining solution, then stained with 50 µl
of 400 mM 7AAD and 50 µl of 100 mM PY for 30
min. The stained cells were analyzed on a flow cytometry using
CellQuest software (Becton Dickinson).
Immunoblotting for MAPKs.
Whole cell lysates (0.5 to 5 x 105 cells/lane) were resolved in 2.5% SDS buffer
and were applied on 12% PAGE and then electroblotted onto
polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). After
blocking with 5% nonfat dry milk in TBST [20 mM Tris (pH
7.4), 150 mM NaCl, and 0.1% Tween 20] for 30 min, the
membranes were probed with the following antibodies: anti-ERK1,
anti-p38 or anti-JNK2 (Santa Cruz, CA), or
anti-phospho(Thr202/Tyr204)-p44/42 MAPK,
anti-phospho(Thr183/Tyr1859)-JNK or anti-phospho(Thr71)-p38 (New
England Biolabs, Schwalbach, Germany). The bound antibodies were
visualized by using Photope-Star Western Blot Detection kit (New
England Biolabs).
Statistical Analysis.
Statistical significance was assessed by Student’s t test,
and P < 0.05 was considered as significant.
Composite treatments were analyzed by Microsoft Excel Software.
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RESULTS |
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. All of the cell lines examined expressed substantial but
varying levels of surface Fas and FasL; Fas expression was highest on
T98G and relatively low on A-172 and YKG-1. FasL expression was highest
on YKG-1, followed by A-172 and T98G. To examine whether the FasL
expressed on these cell lines is functional in mediating cell death,
cytotoxic assay was performed against a Fas-sensitive target cell line
WR19L/Fas (Fig. 1B)
. All these three glioma cells could kill
WR19L/Fas cells, and their cytotoxic activities were correlated with
the levels of FasL expression on these cells. In agreement with a
previous report (8)
, these data indicate that the FasL
expressed on the glioma cells is functional. Next, we sought to
determine whether Fas and FasL may be involved in tumor cell growth
in vitro. For this purpose, we exogenously added an
agonistic anti-Fas mAb (CH-11) to trigger a Fas-mediated signal or a
neutralizing anti-FasL mAb (NOK-2) to block endogenous Fas-FasL
interaction and estimated proliferation by
[3H]TdR incorporation.
[3H]TdR incorporation of YKG-1 cells was
substantially augmented by CH-11 and conversely inhibited by NOK-2
(Fig. 1C)
. This unexpected observation suggested that glioma
cells may use endogenous Fas/FasL for their own growth in an autocrine
manner. We examined whether this augmented
[3H]TdR incorporation after the CH-11 treatment
results in actual increase in cell number; however, the addition of
CH-11 did not result in either increase or decrease of net live cell
counts in glioma cell lines (not shown). These results prompted us to
speculate that Fas may mediate both growth-promoting and death-inducing
signals in glioma cells, and these opposite effects offset each other
and thus resulted in seemingly no change in live cell counts.
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. To assess the effect of CH-11 on the growth of Fas
transfectants, we initially estimated the viability of the
transfectants after 24-h culture with various doses of CH-11 by a
modification of the MTT assay. As shown in Fig. 2B
,
viability of all of the transfectants examined were substantially
reduced by CH-11 at >0.1 µg/ml. In contrast, vector controls did not
show such a reduced viability, and rather a slight increase was noted
in T/M1 and Y/M1. No difference was observed between the vector
controls and the untransfected parental cell lines (not shown). These
results indicate that as a result of Fas overexpression, the
transfectants might gain increased sensitivity to cell death. To
directly determine the Fas-induced cell death, change in mitochondrial
membrane potential (m) and plasma membrane integrity were
simultaneously analyzed by flow cytometry after dual staining with
Rh123 and PI. As shown Fig. 2C
, CH-11 did induce cell death
in transfectants as detected by an increase in PI-positive dead cells
and Rh123-dim-positive apoptotic cells with reduced m
(24)
. To examine whether this reduction of cell viability
might result in a decrease of cell number, we determined net live cell
counts by trypan blue exclusion. As shown in Fig. 3
, net live cell numbers of transfectants A/FF222 and T/FF14 at 24–72 h
were not significantly different regardless of the presence or absence
of CH-11. Although the growth of Y/FF014 was significantly suppressed
by CH-11, it was notable that live cell counts were still increasing
from the initiation of culture. In a marked contrast, the addition of
CH-11 to Jurkat T cells, a representative Fas-sensitive cell line,
resulted in a progressive loss of live cells, and virtually no live
cells could be detected by 72 h. These results indicated that
Fas-expressing glioma cells still grow in the presence of CH-11 while a
substantial part of cells are constantly depleted by Fas-mediated
apoptosis. We interpreted these paradoxical results to suggest that Fas
might promote proliferation of glioma cells, which surpassed the
induced rate of apoptosis and thus resulted in an overall increase of
cell number.
|
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, serum starvation for 24 h could successfully arrest
almost all A/FF222 cells at the G0 phase (as
defined by diploid DNA with basal RNA expression). The serum
deprivation per se did not significantly affect cellular
viability of glioma cells, except that Y/FF014 showed slight
decrease in viability, for the culture period up to 96 h (Table 1
and not shown). In this condition, the CH-11 treatment for 24 h
did result in cell cycle progression as manifested by the emergence of
cells at the S-G2-M phase of the cell cycle, with
high levels of induced RNA expression and simultaneous emergence of
cells with
sub-G0/G1-fragmented DNA
content indicative of apoptotic cells (Fig. 4B)
. As
summarized in Table 1
, all three parameters, the emergence of apoptotic
cells, the S-phase entry, and the induction of RNA expression,
were significantly driven by CH-11 in all of the transfectants derived
from three glioma cell lines. Collectively, these results clearly
indicated that Fas not only mediates apoptosis but also drives cell
cycle progression.
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, after treatment with CH-11 for 15 min, a phosphorylated form of ERK
(Lane 2, top), but not of JNK or p38, was detected. As a
positive control, phosphorylated forms of ERK, JNK, and p38 were
detected in the UV-irradiated cells (Lanes 6). We could
detect this phosphorylated form of ERK in the absence of that of JNK
and p38 for a period up to 60 min after CH-11 stimulation (Lane
3). The phosphorylation of ERK by CH-11 was completely inhibited
by a MEK1 inhibitor PD98059 at a concentration of 10
µM (Lane 4). Furthermore, a caspase
inhibitor zD could also block the phosphorylation of ERK (Lane
5).
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, the addition of PD98059 completely blocked the CH-11-driven S-phase
entry and RNA induction but did not affect the apoptosis. The caspase
inhibitor zD blocked the CH-11-induced apoptosis, as expected, and also
inhibited the CH-11-driven S-phase entry and RNA induction. The
addition of control IgM or each inhibitor alone did not affect the cell
cycle progression or the apoptosis. These results suggested that
Fas-mediated activation of the MEK-ERK pathway is closely linked to the
Fas-driven cell cycle progression but not to the induction of
apoptosis. Combined with the immunoblotting data that zD also blocked
the CH-11-induced ERK activation, ERK is placed downstream of
caspase(s) that transmit a cell growth signal by activating the MEK-ERK
pathway in addition to mediating cell death.
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DISCUSSION |
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,
Lane 1), which was associated with the cell cycle arrest in
the G0 phase. Here, we clearly demonstrated that
stimulation of Fas drives cell cycle progression and ERK
phosphorylation in serum-starved glioma cells. The Fas-driven cell
cycle progression and ERK phosphorylation were completely blocked by an
MEK1 inhibitor PD98059. These results indicate that glioma cells use
the MEK-ERK pathway downstream of Fas for proliferation. We believe our
present observation may represent a common feature of gliomas because
similar results were obtained with three different glioma cell lines
and their transfectants. Because activation of JNK and p38 has been reported during the Fas-induced apoptosis in Jurkat T cells (10 , 11 , 16) , our present observation of ERK phosphorylation in the absence of JNK or p38 phosphorylation in anti-Fas-stimulated glioma cells was somewhat unexpected. Ras activation, which potentially leads to ERK activation, has been implicated in the Fas-induced apoptosis in Jurkat cells (32) , but other investigators have observed that Fas cross-linking does not result in ERK activation in this cell line (10 , 33) . In contrast, it was reported that both ERK and JNK were activated after Fas cross-linking in SHEP cells, a human neuroblastoma cell line (14) . Utilization of MAPK subfamilies downstream of Fas thus appears to be divergent, and these differences are at least partly depending on the cell types used. Although we do not formally rule out the possibility that Fas mediates JNK or p38 activation in gliomas, it is worth noting that a p38 inhibitor SB 203580 had no effect on cell cycle progression or apoptosis in the anti-Fas-stimulated glioma cells at concentrations up to 10 µM.4 In any case, the upstream events that connect Fas to ERK activation in glioma cells remains to be clarified.
In contrast to the effect of a MEK1 inhibitor that did not affect apoptosis induction, a caspase inhibitor was found to block both cell cycle progression and apoptosis and also to inhibit the ERK phosphorylation induced by Fas. This indicates that the Fas-mediated caspase activation in glioma cells plays two independent roles for opposite cellular responses; one is to induce apoptosis and another is to act upstream of the MEK/ERK pathway that is linked to cell cycle progression. In this context, it is noteworthy that the Fas-mediated p38/JNK activation was also shown to be blocked by caspase inhibitors (10, 11, 12 , 16 , 33) and that caspase-mediated proteolytic cleavage of several molecules that potentially activate MAPKs have been reported, including PAK2 (15) , MAP kinase kinase 6b (33) , and MAPK/ERK kinase kinase 1 (12) . In SHEP cells, a dominant-negative form of Ras inhibited the Fas-mediated ERK and JNK activation (14) . Identification of the zD-sensitive caspase(s) acting upstream of ERK in glioma cells awaits further investigation, and apparently the cell type-specific regulation of the Ras pathway downstream of Fas is to be the target of further research.
Regarding the differential roles of MAPK subfamilies in regulating growth or death, our results are complementary with those by Xie et al. (34) who have shown that withdrawal of nerve growth factor from rat PC-12 pheochromocytoma cells resulted in sustained activation of JNK and p38 with concurrent inhibition of ERK, which was associated with apoptosis induction. Similarly, it has been shown that, downstream of the T-cell receptor signaling in thymocytes, the MEK1-ERK pathway was linked to positive selection by promoting thymocyte survival and proliferation, whereas the MAP kinase kinase 6-p38 pathway was involved in negative selection by promoting apoptosis (35) . Among the Fas receptor-binding molecules, FADD plays a critical role in initiating cell death process by triggering the caspase cascade, but paradoxically, several lines of evidence have suggested that it may regulate cell proliferation as well: FADD-deficient T cells (36) and dominant-negative FADD-expressing T cells did not proliferate normally in response to mitogens (37) . In light of this knowledge, our findings are unique in that both cell death and cell growth were induced simultaneously in response to signaling via a single receptor. The glioma cells may thus provide a unique cell culture system for elucidating molecular switch mechanism(s) that determine cell death or growth downstream of Fas.
Because little Fas and FasL mRNA could be detected in the normal brain (38) , it was previously thought that the Fas-FasL system might not be operative in the central nerve system. However, subsequent studies have revealed that this system is actively operative in the central nerve system in some pathological conditions. Activated microglia expressed FasL, which in turn may induce Fas-expressing oligodendrocyte death in multiple sclerosis (39) . As shown in this article and reported by others, some glioma cell lines as well as primary astrocytic brain tumors express Fas and/or FasL (8 , 22 , 40) . Our present study extends these observations and provides a strong implication that gliomas use the endogenous Fas-FasL interaction for proliferation. FasL expression in tumors has been implicated as a means to evade antitumor immune response (5 , 6) . Regarding the pathophysiological role of Fas/FasL expression in tumorigenesis, our present study may thus provide an important implication that some tumors may use this system for progression by promoting its own growth as an autocrine growth factor.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grant-in-aids for scientific
research from the Ministry of Education, Science, Sports and Culture of
Japan (to N. O.; Grants 10180206 and 11161209) and from the
Science and Technology Agency of the Japanese Government (to N. O.) and a grant from Core Research for Evolutional Science and
Technology (to H. Y.). 
2 To whom requests for reprints should be
addressed, at Room 665, Department of Retroviral Regulation, Medical
Research Division, Tokyo Medical and Dental University, 1-5-45 Yushima,
Bunkyo-ku, Tokyo 113-8519, Japan. Phone: 3-5803-5160; Fax: 3-3814-7172;
E-mail: oyaizu.mbch{at}med.tmd.ac.jp
3 The abbreviations used are: MAPK,
mitogen-activated protein kinase; mAb, monoclonal antibody; ERK,
extracellular signal-regulated kinase; JNK, c-Jun
NH2-terminal kinase; zD, Z-Asp-CH2-DCB; Rh123,
rhodamine 123; PI, propidium iodide; 7AAD, 7-amino-actinomycine D; PY,
pyronin Y; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; MAP, mitogen-activated protein; MEK, MAPK/ERK kinase.
4 H. Shinohara, unpublished observations.
Received 10/ 4/99. Accepted 1/19/00.
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m; X-axis;
Rh123) and membrane integrity (Y-axis; PI) were analyzed
on a flow cytometry. Values denote the percentage of cells in each
quadrant.
) or 100 ng/ml CH-11 (•). Cells were
harvested at the indicated time points, and net live cell numbers were
counted by trypan blue exclusion. Data are expressed as mean ± SD of three experiments.
P < 0.001 compared to the
CH-11-treated samples.