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J Biol Chem, Vol. 274, Issue 40, 28505-28513, October 1, 1999
From the The Cdc2L locus encoding the PITSLRE protein
kinases maps to chromosome band 1p36 and consists of two duplicated and
tandemly linked genes. The purpose of the present study was to
determine whether diminution of PITSLRE kinases leads to deregulation
of apoptosis. The human melanoma cell lines A375 (Cdc2L wild-type alleles) and UACC 1227 (mutant Cdc2L alleles) were tested with agonist
anti-Fas monoclonal antibody. We found that exposure of these cells to
anti-Fas for 24, 48, or 72 h resulted in differential sensitivity
to Fas-induced apoptosis. In A375, cell death started at 24-48 h
post-treatment, and it was maximal by 72 h. Conversely, UACC 1227 cells were resistant to Fas-mediated apoptosis. Induction of PITSLRE
histone H1 kinase activity was observed in A375 anti-Fas treated but
not in UACC 1227 cells. Also, the PITSLRE protein kinase activity in
A375 anti-Fas-treated cells preceded maximal levels of apoptosis.
Finally, fluorescence confocal microscopy revealed a nuclear
localization of PITSLRE proteins in normal melanocytes and A375 cells
but a cytoplasmic localization in UACC 1227 cells. The differences in
PITSLRE protein and cellular localization between A375 and UACC 1227 cells appear to account for the differences in sensitivity of the two
cells lines to anti-Fas and staurosporine. These observations suggest
that alterations in PITSLRE gene expression and protein localization
may result in the loss of apoptotic signaling.
Apoptosis is a highly regulated process that plays a major role in
development and homeostasis (1). The pathways of cellular proliferation
and apoptosis appear to be linked to minimize the occurrence of
neoplasia (2). It has also been proposed that deregulation of apoptosis
is a pathogenic process in some bone marrow disorders (3) and in tumor
development (4, 5). The cell surface receptor Fas/APO-1 (CD95) is a
type-I transmembrane protein that belongs to the tumor necrosis factor
(TNF)1 and nerve growth
factor (NGF) receptor superfamily (6-8). Binding of anti-Fas antibody
or Fas ligand (FasL) to Fas receptor triggers apoptosis in
vivo and in vitro, in sensitive cells (8, 9). There is
evidence suggesting that apoptosis induced by TNF and Fas involves a
common mechanism. Both Fas and TNF receptors contain dead domains (DD),
which provide receptor-triggered signaling that may allow
"cross-talk" between their pathways (10-12). TNF and Fas-mediated
apoptosis involve a family of cysteine proteases related to the
interleukin-1 Malignant melanoma is a relatively common neoplasm and the only
cutaneous malignancy that metastasizes and causes death. The incidence
of melanoma is rising faster than any other cancer in the United
States, and it is expected to reach an all time high rate of 1 in 75 by
the year 2000. Recent evidence suggests that failure of cells to
undergo apoptotic cell death might contribute to the pathogenesis of a
variety of human diseases including cancer (18). Previous work in our
laboratory demonstrated that deletions of chromosome region 1p36 are
one of the most frequent cytogenetic abnormalities found in melanoma
(19). The Cdc2L locus encoding the PITSLRE protein kinases maps to
chromosome band region 1p36 (20). We have also shown that one allele of
the Cdc2L locus on 1p36 was either deleted or translocated in eight of
fourteen different melanoma cell lines (21). Decreased expression of the PITSLRE proteins from the remaining allele was observed in several
cell lines and surgical malignant melanoma specimens (21).
The PITSLRE proteins are part of the large family of
p34cdc2-related kinases whose functions appear
to be linked to control of cell division and possibly programmed cell
death (22-25). The PITSLRE p110 isoforms are reported to be involved
in the regulation of RNA splicing/transcription during the cell cycle
(26). The larger p110 PITSLRE isoforms are also cleaved by
multiple caspases during Fas- and TNF Cell Culture--
A375 and UACC 1227 human melanoma cell lines
were obtained from the Arizona Cancer Center Tissue Culture Shared
Resource. Human melanoma cells were grown as monolayers in RPMI 1640 medium supplemented with 5% (v/v) dialyzed and heat-inactivated fetal calf serum, 1% L-glutamine, and 1%
penicillin-streptomycin (10,000 units/ml-10,000 µg/ml). Normal human
melanocytes were isolated from newborn foreskin and cultured in
modified M15 medium supplemented with 5% fetal calf serum as described
previously (37). Media and reagents were purchased from Life
Technologies, Inc., Grand Island, NY.
Cell Surface Expression of Fas--
Cell surface expression of
Fas receptor in A375 and UACC 1227 melanoma cells was detected by flow
cytometry (38). Briefly, cells were harvested, washed twice with PBS
and incubated for 60 min on ice with 20 µg/ml of either anti-Fas
mouse monoclonaI IgM antibody or a nonspecific isotype-matched
monoclonal antibody in PBS containing 1% fetal calf serum, 0.02 mM NaN3, and 0.5 mM EDTA. All the
chemicals and monoclonal antibodies were purchased from Sigma. Cells
were washed twice with PBS and incubated for 30 min on ice with 10 µg/ml of affinity purified FITC-conjugated goat anti-mouse IgM
(Becton Dickinson). Cells were washed twice with PBS and analyzed for
Fas expression on a FACScan flow cytometer (Becton Dickinson).
RNase Protection Assay--
Total RNA isolated from HeLa cells
(5 µg) and the melanoma cell lines A375, UACC 1227, and UACC 903 was
analyzed for distinct mRNA species using PharMingen's
RiboQuantTM multi-probe with the hAPO-2, hAPO-3, and hAPO-5
probe template sets. Cell Treatment--
On day 1, A375 and UACC 1227 cells (1 × 106) were cultured in RPMI 1640 medium supplemented with
5% (v/v) dialyzed and heat-inactivated fetal calf serum, 1%
L-glutamine, and 1% penicillin-streptomycin and incubated
overnight at 37 °C in a humidified 5% CO2 environment. On day 0, cells were washed twice with PBS and treated with either 0.5 µg/ml anti-Fas monoclonal antibody, CH-11 (anti-Fas mAb, Upstate Biotechnology, Lake Placid, NY) or 10 ng/ml staurosporine for 24, 48, and 72 h at 37 °C. Anti-Fas mAb-treated and untreated control
cells were harvested by low speed centrifugation and washed twice with
PBS for staining with 7-amino-actinomycin D (7AAD) and flow cytometric analysis.
Flow Cytometric Analysis--
Anti-Fas mAb or
staurosporine-treated and nontreated control cells (1 × 106) were stained with 7AAD (200 µg/ml, Sigma) in PBS and
incubated for 20 min at 4 °C in the dark (28). Cells were harvested
by low speed centrifugation, resuspended in PBS, and analyzed for apoptosis using a FACStar flow cytometer (Becton Dickinson). Unstained A375 and UACC 1227 cells were used as negative controls. Discrimination of the three populations (dead cells as 7AAD-bright, apoptotic cells as
7AAD-dim, and live cells as 7AAD-negative) was validated by cell
sorting and morphological examination.
Cell Morphology--
Cytospin preparations of anti-Fas mAb and
staurosporine-treated and nontreated control cells (1 × 105) from unsorted and FACStar sorted populations were
stained using the DiffQuik (Baxter) staining method. Briefly, slides
were fixed (1.8 mg/liter triacylmethane dye and 100% PDC (0.625 g/liter azure A and 0.625 g/liter methylene blue in methyl alcohol))
for 30 s, stained in solution I (1 g/l xanthene and 100% PDC) for
1 min, and then in solution II (1.25 mg/liter triazine dye mixture,
100% PDC) for 1 min. Slides were air-dried and analyzed by light
microscopy at 100×.
MTT Assay--
A375 and UACC 1227 cell viability was assessed
using the MTT assay. Cells (2 × 104/well) were plated
in 96-well microtiter plates in the presence of fresh RPMI, 5% fetal
bovine serum medium. The next day, cells were incubated with either
staurosporine (0.5-100 ng/ml) or anti-Fas (500 µg/ml) and caspase 8 inhibitor, caspase 3 inhibitor, or both inhibitors for 24, 48, or
72 h at 37 °C. MTT (2 mg/ml in PBS) was added to the wells
following cell treatment and the incubation continued for 4 h at
37 °C. The precipitate was eluted with 100% Me2SO at
room temperature for 10 min and optical density values were measured at
540 nm, using the Biomek plate reader. Survival was expressed as the
percentage of viable cells in treated samples relative to nontreated
control cells.
Histone H1 Kinase Assay--
A375 and UACC 1227 cells were
treated with anti-Fas mAb for 24, 48, and 72 h. A375 cells were
also treated with anti-Fas, staurosporine (10 ng/ml), or in combination
with protease inhibitors of caspase 3 (DEV-FMK, 20 µM) or
caspase 8 (IETD, 20 µM) for 48 h, harvested, washed
twice with ice-cold PBS and lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 5 mM EGTA, 15 mM
MgCl2) containing 60 mM Western Blot Analysis--
Western blot analysis of various
melanoma cell lines were performed using the PITSLRE P2N100 (1:5000)
affinity purified polyclonal antisera, PARP (1:1000) or
Microinjection and Transfection--
Cells were grown on 35-mm
tissue culture dishes containing sterile glass coverslips embedded in
the plastic (Martek Corporation). Prior to microinjection, the plasmid
DNAs (pCH110, pcDNA 3.0, and DR3/pcDNA 3.0) were diluted to 200 ng/µl in 50 mM Hepes, 100 mM KCL, 40 mM Na2PO4, pH 7.2, and the pCHO110
reporter plasmid was mixed with an equal volume of either the control
(pcDNA 3.0) or the test plasmid (DR3/pcDNA 3.0). For each
experiment, identical number of cells in three different areas of the
dish were injected with the automatic Eppendorf Transjector II system
using a femtotip II capillary, with an injection pressure of 59 p.s.i. and an injection time of 0.6 s. After injection, the cells
were returned to the 37 °C incubator. Sixteen hours later, the cells
were stained for Immunofluorescence Confocal Microscopy--
Normal melanocytes,
A375 and UACC 1227 melanoma cells, were grown on coverslips, washed
twice with PBS and fixed in formalin for 20 min at room temperature
(RT). Cells were rinsed three times with PBS and permeabilized with
100% methanol at Statistical Analysis--
Statistical analyses on time course of
anti-Fas mAb-induced apoptosis in melanoma cell lines were performed
using the standard Student's t test.
Human Malignant Melanoma Cell Lines Express Fas Receptor--
To
rule out the possibility that A375 or UACC 1227 cells are resistant to
anti-Fas mAb-mediated cell death because they do not express Fas
receptor, cells were analyzed for the expression of cell surface Fas
receptor by immunofluorescence and flow cytometry. Immunofluorescence
and flow cytometric analysis demonstrated that both cell lines, A375
and UACC 1227, express quantitatively similar levels of Fas receptor on
the cell surface (Fig. 1). Furthermore, confocal microscopy revealed that the Fas receptor was localized on the
cell membrane as well as, in the cytoplasm (data not shown). In
addition, RNase protection analysis indicated that both cell lines
express all of the components involved in the Fas/DR3/TNF signal
transduction pathway(s) (Fig. 2,
A and B). Although mRNA levels for Fas
receptor were decreased in UACC 1227 cells when compared with A375 by
RNase protection assay (Fig. 2A), UACC 1227 cells express
similar or higher levels of cell surface Fas receptor than A375 by flow
cytometric analysis (Fig. 1). This suggests that the efficiency of
translation of CD95 mRNA in UACC 1227 cells is higher than that of
A375 cells.
Anti-Fas mAb Treatment Induces Apoptosis in A375 Cells but Not in
UACC 1227 Cells--
It has been reported that PITSLRE kinases might
serve as effectors of an apoptotic signaling pathway(s) (25). To test
this hypothesis, we used the melanoma cell lines A375, which has normal PITSLRE alleles and exhibits normal expression of PITSLRE, and UACC
1227, which has an abnormal PITSLRE allele and exhibits decreased PITSLRE expression (21). To determine the effect of PITSLRE expression
on apoptotic signaling, flow cytometric analysis using 7AAD was
performed. 7AAD is a fluorescent DNA-binding agent that intercalates
between cytosine and guanine bases, and it is used to detect dead
(7AAD-bright), apoptotic (7AAD-dim), and live (7AAD-negative) populations by fluorescence-activated cell sorting (28). Apoptosis was
triggered using anti-Fas mAb CH-11. Exposure of the cells to anti-Fas
mAb for 24, 48, or 72 h demonstrated that Fas-induced apoptosis
begins in A375 cells 24-48 h post-treatment (5-23% apoptotic cells)
and is maximal by 72 h (80% apoptotic cells) (Fig.
3, A and B).
Conversely, UACC 1227 cells were resistant to Fas-induced apoptosis
(Fig. 3B). The 7AAD data was validated by 1) cell sorting and morphological examination using Wright/Giemsa (DiffQuik staining method, 2) terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL), and 3)
hematoxylin and eosin staining. Morphological changes consistent with
apoptotic cell death including cell shrinkage, nuclear condensation, and membrane blebbing were observed in the Fas-sensitive A375 cells
(Fig. 3C, a and b). In contrast, no
morphological changes were seen in the UACC 1227 cells (Fig.
3C, c and d). Morphological analysis
of sorted A375 cells treated with anti-Fas mAb for 72 h indicated
that cells from early-late/dead apoptotic regions show nuclear
condensation with marked cell shrinkage (data not shown). These data
demonstrate that A375 cells are sensitive to Fas-mediated apoptosis,
whereas the UACC 1227 cells are resistant to Fas-induced apoptosis.
Because both cell lines have Fas receptor and all of the components of
the Fas-mediated signal transduction pathway, these differences may
reflect the involvement of PITSLRE kinases in the Fas-mediated
signaling pathway in melanoma cells.
Processing of PITSLRE Proteins and Activation of PITSLRE Kinase
Activity in Melanoma Cell Lines during Anti-Fas mAb-mediated
Apoptosis--
It is not known what substrate is responsible for the
execution of the death sentence once the process of apoptotic cell
death has been activated. It has been suggested that PITSLRE kinases might be potential candidates (14) because they are processed and
activated following anti-Fas treatment in T cells (25). However, it is
not known whether processing and activation of PITSLRE kinases take
place in melanoma cells. To determine whether PITSLRE kinases are
activated in melanoma cells following treatment with anti-Fas mAb, A375
and UACC 1227 cells (1 × 106) were exposed to
anti-Fas mAb for 24, 48, or 72 h, and PITSLRE kinase activity was
measured by the histone H1 kinase assay. After 48 h of treatment
with anti-Fas mAb, PITSLRE kinase activity was increased 8-fold in A375
cells, which returned to control levels by 72 h (Fig.
4A). Cleavage of PITSLRE
proteins and poly (ADP-ribose) polymerase (PARP) was observed in A375
cells treated with anti-Fas mAb as early as 24 h post-treatment,
and it reached maximum levels by 48 h after treatment (Fig.
5). The maximal cleavage level of PITSLRE
and PARP occurred at 48 h following anti-Fas treatment and
correlates with the maximum activation levels of PITSLRE kinase activity. Conversely, there was no increase in PITSLRE kinase activity
in UACC 1227 Fas-treated cells at any of the time points analyzed (Fig.
4B). Furthermore, no cleavage of PITSLRE or PARP was
observed in UACC 1227 cells treated with anti-Fas (data not shown).
These results are consistent with the apoptosis data presented above
and show that PITSLRE kinase activity precedes maximal levels of
apoptosis.
Nuclear Microinjection of DR3 in A375 and UACC 1227 Melanoma
Cells--
Although the RNase protection experiments indicated that
the downstream components of the Fas pathway were present in these cells, it was necessary to determine whether they were functional. One
approach to this question was to examine the sensitivity of the two
cell lines to signaling events mediated by other death receptors, such
as DR3, or to agents that bypass the receptor pathway, such as
staurosporine. We reasoned that if the two cell lines were equally
sensitive to the DR3 death receptor cross-linking, the Fas receptor
itself may be defective. Differences in staurosporine sensitivity would
be more likely to reflect alterations in function of other downstream
components of the pathway. Therefore, equal amounts of
A375 and UACC 1227 Cells Differ in their Sensitivity to
Staurosporine-induced Apoptosis--
To determine whether alternative
apoptotic pathways exist in UACC 1227 cells, staurosporine was used.
Staurosporine is a death inducer known to cause apoptosis through
cytochrome c release from the mitochondria and activation of
caspase 9 (29, 30). A dose-response curve was performed to determine
the optimal staurosporine concentration to be used for the following
experiments (Fig. 6A). A
staurosporine concentration of 10 ng/ml, which caused a 50% decrease
in A375 cell viability, was used for subsequent experiments. Exposure
of A375 and UACC 1227 cells to staurosporine (10 ng/ml) for 24, 48, or
72 h demonstrated that staurosporine-induced apoptosis begins in
A375 cells at 24 h post-treatment (50% apoptotic cells), and it
is maximal by 72 h (70% apoptotic cells) (Fig. 6B).
Conversely, UACC 1227 cells were resistant to staurosporine-induced
apoptosis at this concentration (Fig. 6B). Morphological
changes consistent with apoptotic cell death were observed in the
staurosporine-sensitive A375 cells (Fig. 6C, a
and b). In contrast, the same concentration of staurosporine
did not produce morphological changes in UACC 1227 cells (Fig.
6C, c and d). A 10-fold increase in
the concentration of staurosporine (100 ng/ml) was required to induce
the same percentage of apoptotic cells in UACC 1227 as that observed in
A375 cells treated with 10 ng/ml (Fig. 6A). These results
demonstrate that A375 cells are more sensitive to staurosporine-induced
apoptosis than UACC 1227 and that an alternative apoptotic pathway is
operational in UACC 1227 cells.
Caspase 3 and Caspase 8 Inhibitors Block Fas-mediated Cell Death in
Melanoma Cells--
To determine whether caspases 3 and/or 8 are
involved in Fas-induced apoptosis in melanoma, A375 cells were
pre-incubated with protease inhibitors of caspase 3 (DEV-FMK, 20 µM), caspase 8 (IETD, 20 µM), or both for
3 h, and their effect on cell survival following Fas treatment for
24, 48, or 72 h was examined. As shown in Table
I, both inhibitors blocked Fas-mediated
cell death, suggesting that both caspases 3 and 8 are involved in the
Fas signaling pathway in melanoma cells.
Stimulation of PITSLRE Kinase Activity in A375 Cells following
Treatment with Anti-Fas and Staurosporine Is Markedly Reduced by
Protease Inhibitors of Caspases 3 and 8--
To further study the
involvement of PITSLRE kinases in anti-Fas- and staurosporine-mediated
apoptosis, we examined whether protease inhibitors of caspases 3 and 8 had any effect on preventing the stimulation of PITSLRE kinase activity
during anti-Fas- or staurosporine-induced apoptosis. A375 cells were
exposed to the apoptosis-triggering stimuli (anti-Fas mAb or
staurosporine) alone or in combination with inhibitors of caspases 3 and 8, and PITSLRE kinase activity was measured by the histone H1
kinase assay. As shown in Fig. 7, both
inhibitors markedly reduced (50-60%) anti-Fas and staurosporine
activation of PITSLRE kinase activity following simultaneous treatment
with the inhibitors (Fig. 7B). We also demonstrated that
exposure of A375 cells to the apoptosis-inducing agent staurosporine
for 48 h resulted in the stimulation of PITSLRE kinase activity
(Fig. 7). These observations suggest that caspases 3 and 8 play an
important role in anti-Fas- and staurosporine-mediated activation of
PITSLRE kinase activity during apoptosis.
Cellular Localization of PITSLRE p110 Isoforms in Human Melanoma
Cells--
PITSLRE p110 isoforms are ubiquitously expressed in
proliferating cells. However, the localization of p110 PITSLRE isoforms and their function(s) in normal and transformed melanoma cells are
unknown. To determine whether there were differences in the cellular
localization of PITSLRE isoforms in the melanoma cell lines A375 and
UACC 1227 relative to normal melanocytes and whether there is a link
between the localization of PITSLRE isoforms and apoptotic signaling,
immunofluorescence confocal microscopy analysis was performed. The
results demonstrate that p110 PITSLRE isoforms localize to the nucleus
in normal melanocytes and A375 cells, as detected by using the PITSLRE
specific antibody P2N100 which recognizes the p110 Previous studies in our laboratory using fluorescence in
situ hybridization indicated that one allele of the PITSLRE gene complex on chromosome 1 was either deleted or translocated in several
melanoma cell lines. Furthermore, the expression of PITSLRE proteins
from the remaining allele was decreased in several melanoma cell lines
and surgical melanoma specimens (21). Similar results have been
observed in neuroblastoma and in childhood endodermal sinus tumors
(20). However, the functional consequences of genetic alterations
within the Cdc2L locus encoding the PITSLRE kinases in regards to the
development of melanoma are not known. Because the PITSLRE p110
isoforms may be involved in apoptosis, we wanted to evaluate the
functional consequences of PITSLRE gene alterations with regard to
Fas-mediated apoptotic signaling in melanoma cell lines.
In the present study, we provide evidence that the melanoma cell lines
A375 and UACC 1227 express Fas receptor and that anti-Fas mAb induces
apoptosis in A375, but not in UACC 1227 cells. Morphological changes
consistent with apoptosis including cell shrinkage, nuclear condensation, and membrane blebbing were observed in the A375 Fas-sensitive cells, but not in the UACC 1227 Fas-resistant cells.
We demonstrate that the A375 melanoma cells, which have normal Cdc2L
alleles and normal PITSLRE protein expression, are sensitive to
Fas-induced apoptosis. In contrast, UACC 1227 cells, which have
decreased PITSLRE expression and mutant alleles, do not undergo Fas-induced apoptosis. In addition, an increase in PITSLRE kinase activity was observed in A375 Fas-sensitive cells, but not in the UACC
1227-resistant cell line. Stimulation of PITSLRE kinase activity was
also observed in A375 cells following staurosporine treatment. The
stimulation of PITSLRE kinase activity was markedly reduced (50-60%)
by caspase inhibitors DEV-FMK or IETD during Fas- and
staurosporine-mediated cell death. We also report the caspase cleavage
of PITSLRE protein and PARP during Fas-induced apoptosis. These
observations demonstrate that PITSLRE kinase activation is associated
with Fas- and staurosporine-mediated apoptosis in melanoma cells.
Furthermore, the data presented here suggest that multiple caspases
appear to be involved in the cleavage of PITSLRE during Fas-induced
apoptosis in A375 cells. Recently, it has been reported that PITSLRE
kinases are specifically cleaved in response to TNF by caspases 1 and 3 resulting in the activation of the PITSLRE kinase, both in
vivo and in vitro (27, 31). However, the proteases
responsible for the processing and activation of PITSLRE kinases in
melanoma and the importance of this processing in apoptosis are
unknown. We also demonstrate that caspases 3 and 8 are involved in the
Fas signaling pathway in melanoma cells, and caspase 3 is involved in
staurosporine-mediated cell death, which is consistent with the
published reports on PITSLRE p110 isoforms being cleaved and activated
by caspases during apoptosis (27, 31).
Finally, in this study we demonstrate that there is a difference in the
subcellular localization of p110 PITSLRE isoforms in UACC 1227 melanoma
cells relative to normal melanocytes. This result suggests that UACC
1227 cells express PITSLRE proteins that either lack the nuclear
translocation signal or that contain a point mutation affecting this
region of the protein. The amino-terminal domain also contains several
distinct regions that may specify nuclear localization and protein
stability (32, 33). Current studies in our laboratory, involving
polymerase chain reaction-SSCP and direct DNA sequence analysis,
suggest that UACC 1227 cells have a mutation(s) in the nuclear
localization signal, which may explain the abnormal cytoplasmic
localization of p110 PITSLRE isoforms. Loss or inactivation of the
nuclear translocation signal in UACC 1227 cells and consequent
cytoplasmic localization of p110 PITSLRE isoforms. In addition,
resistance to Fas suggests a functional role of PITSLRE protein kinases
in mediating Fas-induced apoptosis. Furthermore, if we consider a role
for PITSLRE p110 isoforms in Fas-mediated signal transduction
processes, altered distribution of PITSLRE kinases in transformed cells
may contribute to the transformed phenotype by deregulating the
processes of apoptosis, spliceosome formation, or assembly/disassembly
of nuclear speckles, which is involved in the regulation of RNA
splicing/transcription (26).
A question that remains unknown is the identity of the substrate that
executes the final death sentence following activation of caspases. One
candidate (or family of candidates) is the PITSLRE kinases. PITSLRE
kinase proteins have been shown to be processed and activated in cells
treated with anti-Fas and TNF (25, 27, 31). Circumstantial evidence
suggests that, following the processing and activation of PITSLRE
kinases, they may be released from associated restraining molecules to
execute the final death sentence. However, this important role for the
PITSLRE kinases as final executioners of apoptosis needs to be further
investigated. The mechanism that we are proposing to explain the role
of PITSLRE protein kinases during apoptosis in malignant melanoma cells
is shown in Fig. 9. Activation of Fas
receptor by anti-Fas monoclonal antibody results in the aggregation and
rapid recruitment of FADD (11). The interaction of FADD and Fas through
their carboxyl-terminal death domains unmasks the amino-terminal death
effector domain of FADD, allowing it to recruit and activate
pro-caspase-8 to the Fas signaling complex (34), Caspase 8 activates
pro-caspase 3 either directly (pathway 2) or indirectly through
cytochrome c release from the mitochondria (pathway 1).
Cytochrome c forms a complex with apoptotic protease
activating factor 1 (Apaf-1) that binds and activates pro-caspase 9 (35). Activated caspase 9 binds to and activates pro-caspase 3. Our
data indicate that PITSLRE kinase activation precedes Fas-induced
apoptosis, and it is a downstream event. Because PITSLRE kinases have
cleavage sites for caspases 3 and 8 and it has been shown that PITSLRE p110 isoforms are cleaved by these caspases during Fas-induced apoptosis in Jurkat cells, we are proposing that PITSLRE kinases get
cleaved and activated by caspases 3 and 8 in melanoma cells. Activation
of PITSLRE protein kinase results in the phosphorylation and activation
of unknown downstream substrates and subsequent transcription of genes
involved in the final stages of apoptosis. Staurosporine-induced cell
death is also another operational apoptotic pathway present in
malignant melanoma cells and is shown in Fig. 9. The fact that
staurosporine-induced cell death, which bypasses receptor-mediated
signaling, is altered in UACC 1227 cells suggests that PITSLRE kinases
are involved in the staurosporine death signaling pathway. Furthermore,
these data demonstrate that PITSLRE protein kinases are involved in
Fas-, staurosporine-, and DR3-mediated cell death signaling pathways.
However, the function of these PITSLRE kinases during apoptosis is not
known.
Finally, the data presented here suggests that alterations in Cdc2L
gene expression and protein localization can result in the loss or
deregulation of apoptotic signaling pathway(s). Therefore, altered
PITSLRE kinases may represent a different mechanism from that reported
for Fas ligand that could contribute to the immune privilege in
malignant melanoma (36). Deregulation of apoptotic signaling pathways
may represent a mechanism to enhance tumorigenesis by preventing the
elimination of these cells through normal checkpoint control.
We acknowledge Dr. Claire Payne for technical
assistance with confocal microscopy.
*
This research was supported by a grant from National
Institutes of Health (R29-CA 70145-01), by the friends of the Arizona Cancer Center, by a grant from National Institutes of Health (GM44088) (to V. J. K.) and by the SWEHSC Core Grant P30ES06694.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.
The abbreviations used are:
TNF, tumor necrosis
factor;
CHO, Chinese hamster ovary;
PBS, phosphate-buffered saline;
FITC, fluorescein isothiocyanate;
mAb, monoclonal antibody;
7AAD, 7-amino-actinomycin D;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
DDT, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane;
X-gal, 5-bromo-4-chloro-3-indolyl
Fas-induced Apoptosis in Human Malignant Melanoma Cell Lines
Is Associated with the Activation of the
p34cdc2-related PITSLRE Protein Kinases*
§,,§
Arizona Cancer Center, Tucson, Arizona
85724, the § Pathology Department, University of
Arizona, Tucson, Arizona 85724, and the ¶ Department of Tumor
Cell Biology, St. Jude Children's Research Hospital,
Memphis, Tennessee 38101
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-converting enzyme (ICE-like) family, which are
currently considered to be the central executioners of apoptosis
(13-15). Although Fas and its ligand are expressed in a variety of
cells, including melanocytes, their importance in negative growth
regulation has been studied primarily in the immune system (4, 16, 17).
Thus, their role in apoptotic events concerning nonimmune tumor cells
needs to be further investigated.
-induced cell death (27).
Furthermore, ectopic expression of a p50-PITSLRE construct that
resembles the final caspase-modified product induces apoptosis in CHO
cells (25). Finally, Fas-mediated T-cell death is correlated with PITSLRE proteolysis and increased histone H1 kinase activity (25). The
purpose of the present study was to determine whether alterations in
the PITSLRE isoforms could lead to a disruption in the apoptotic signaling pathway(s) in cultured melanoma cells.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P-Labeled antisense RNA probes
were synthesized, allowed to hybridize to target RNA, and digested with
RNases, as described by the manufacturer. The remaining RNase-protected
probes were purified and resolved on denaturing polyacrylamide (5%)
gels at 40 watts for 3 h, dried, and analyzed by autoradiography
(
80 °C, overnight).
-glycerol phosphate,
0.1 mM sodium orthovanadate, 0.1 mM sodium
fluoride, 15 mM p-nitrophenylphosphate,
aprotinin (10 µg/ml), leupeptin (10 µg/ml), soybean trypsin
inhibitor (10 µg/ml), 1 mM phenylmethylsulfonyl fluoride,
and 0.1 mM benzamide for 30 min. Following lysis, cells were centrifuged at 14,000 rpm for 10 min at 4 °C and resuspended in
lysis buffer, and the protein content was determined using the
bicinchoninic acid assay (Pierce) with bovine serum albumin as the
standard. Total cell lysate (200 µg) was pre-cleared with rabbit sera
to mouse, and PITSLRE proteins were immunoprecipitated using PITSLRE
GN1 affinity purified polyclonal antisera directed against the first 72 amino acids of the p58 PITSLRE kinase 1 (39) and protein A-agarose.
The resultant immunoprecipitates were analyzed for histone H1 kinase
activity using H1 buffer (50 mM Tris-HCl (pH 7.5), 15 mM MgCl2, 1 mM DDT), 50 µM ATP, 8 µCi [32P]ATP (>3000
Ci/mM), and histone H1(2-5 µg/µl). Histone H1
phosphorylation was analyzed by 15% SDS-polyacrylamide gel
electrophoresis and autoradiography. The gels were exposed either for
4 h or overnight at 80 °C on Kodak X-AR5 film. Quantitation
of histone H1 phosphorylation by PITSLRE kinase was determined by
phosphoimaging. The gels were visualized with a Molecular Dynamics 400A
PhosphorImagerTM, and the relative kinase activity was
estimated by quantitating the labeled histone H1 bands using the
Molecular Dynamic ImageQuant software.
-actin(1:8000) antibodies as described previously (21, 31). Briefly,
protein extracts (either 30 or 50 µg) from control and treated
samples were separated by SDS-polyacrylamide gel electrophoresis
transferred to a polyvinylidene difluoride membrane and the blots
probed with the different antibodies. A secondary probe with
horseradish peroxidase-labeled antibodies (Amersham Pharmacia Biotech)
was detected by enhanced chemiluminescence (ECL) detection reagents
(Amersham Pharmacia Biotech).
-galactosidase activity. After an overnight
incubation, the blue cells in each field were quantitated by visual
inspection. For the transfection experiments, 5 × 105
cells were seeded into three 35-mm dishes. Following an overnight incubation, the cells were transfected using the Fugene reagent (Roche
Molecular Biochemicals) according to the protocol of the manufacturer.
Hours (24 h) after transfection, the cells were harvested and stained
with X-gal as described previously. The number of X-gal-stained cells
in 20 random fields were quantitated for each dish.
20 °C for 6 min. Cells on coverslips were
incubated with 5% bovine serum albumin in PBS for 10 min and removed.
Goat serum (1:10 dilution in PBS) was added to all coverslips for 10 min, removed, and then the coverslips incubated with the primary
antibody P2N100 (1:500 dilution) directed against PITSLRE proteins for
1 h at RT. Coverslips were washed three times with PBS for 5 min
each and then incubated with streptavidin (1:100) for 30 min at RT.
Following incubation, coverslips were rinsed three times with PBS for 5 min and incubated with biotin for 30 min. Coverslips were washed three
times with PBS for 5 min, and biotinylated goat anti-rabbit (GAR, 1:100
dilution with 1% bovine serum albumin in PBS) was added for 1 h.
Coverslips were washed again with PBS and incubated with
Cy5-streptavidin for 1 h, washed three times with PBS, and
incubated with RNase (100 µg/ml) for 1 h. Following incubation,
coverslips were washed three times and incubated with YoYo-1 (1:50
dilution) for 15 min. Finally, coverslips were washed with PBS for 15 min, mounted with DAKO mounting media, and store at 4 °C overnight
for immunofluorescence confocal microscopy analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (25K):
[in a new window]
Fig. 1.
Detection of Fas receptor expression, in A375
and UACC 1227 melanoma cells, by flow cytometry. A375 and UACC
1227 cells were stained with anti-Fas mAb or isotype-matched control
and analyzed on a FACScan flow cytometer. The data are presented as the
peak fluorescence intensity (log scale) of cells stained with
isotype-matched control or anti-Fas antibody.
View larger version (70K):
[in a new window]
Fig. 2.
Expression of proteins involved in the
Fas/TNFR/DR3 signal transduction pathways in melanoma cells by RNase
protection analysis using the hAPO-3 (A), hAPO-5
(B), and hAPO-2 (C) probe template
sets.
View larger version (33K):
[in a new window]
Fig. 3.
Fas-induced apoptosis in melanoma cells.
A, scattergrams of 7AAD-stained cells. Untreated control
A375 cells and A375 cells treated with 0.5 µg/ml anti-Fas mAb for 24, 48, and 72 h. FSC, forward light scatter;
FL3, 7AAD fluorescence; Apop, apoptotic;
R1, live cells; R2, early apoptotic cells;
R3, late apoptotic and dead cells. B, time course
of anti-Fas mAb-induced apoptosis in melanoma cell lines. A375 and UACC
1227 cells were treated with anti-Fas mAb (0.5 µg/ml) for 24, 48, or
72 h, stained with 7AAD, and analyzed for apoptosis by flow
cytometry. Results are expressed as the mean percentage of apoptotic
cells ± S.D. of at least three experiments (p < 0.005). C, morphological analysis of A375 and UACC 1227 cells treated with anti-Fas mAb (0.5 µg/ml) for 72 h.
Magnification ×100. a, A375 nontreated cells; b,
A375 cells treated with anti-Fas; c, UACC 1227 nontreated
cells; d, UACC 1227 cells treated with anti-Fas mAb.
View larger version (34K):
[in a new window]
Fig. 4.
Stimulation of PITSLRE kinase(s) activity
during anti-Fas mAb-induced apoptosis in melanoma cells. Histone
H1 was used as the substrate for the assays. Shown is an autoradiograph
of histone H1 kinase activity from control and anti-Fas mAb cell
extracts of A375 (A) and UACC 1227 (B).
Con. 24, 24-h nontreated control; Fas 24, 24-h
anti-Fas; Con. 48, 48-h control; Fas 48, 48-h
anti-Fas; Con. 72, 72-h control; Fas 72, 72-h
anti-Fas. Relative kinase activity from the phosphorylated histone H1
band was determined by phosphoimaging, as described under "Materials
and Methods."
View larger version (88K):
[in a new window]
Fig. 5.
PITSLRE proteins and PARP get cleaved during
Fas-induced apoptosis. Protein extracts (30 µg) from control and
Fas (0.5 µg/ml) treated A375 cells were separated by
SDS-polyacrylamide gel electrophoresis (4-20% gel), transferred to a
polyvinylidene difluoride membrane and assessed for PITSLRE and PARP
cleavage at the indicated times, using PITSLRE P2N100 and actin
antibodies.
-galactosidase plasmid, and either pcDNA 3.0 (a control plasmid)
or a DR3/pcDNA 3.0 expression construct were microinjected into the
nucleus of the A375 and UACC 1227 cells. Sixteen hours later, the cells
were analyzed for X-gal activity. The number of X-gal positive A375
cells declined by 98% when the DR3 expression construct was
co-injected. Conversely, 64% of UACC 1227 cells co-injected with the
reporter, and DR3 plasmids underwent cell death. To confirm these
studies, the two cell lines were also transfected with the same
expression constructs. The transfection results for the A375 cells were
identical to those obtained in the microinjection studies, whereas the
UACC 1227 cells were slightly less sensitive (46% survival). A portion
of this difference may be because of the lower expression level of the
transfected DNA, as judged by the intensity of the X-gal staining. Even
so, there was a significant difference in the ability of these cell
lines to undergo DR3-induced apoptosis. Because DR3 expression studies suggested that there might be differences in the ability of the two
cell lines to respond to death-receptor-mediated signaling events, we
wanted to determine whether the two cell lines also differed in their
responses to apoptosis-inducing agents, such as staurosporine, that do
not require a functional death receptor pathway.
View larger version (33K):
[in a new window]
Fig. 6.
Staurosporine-induced apoptosis.
A, dose-response curve of staurosporine-induced cell death
in A375 and UACC 1227 cells for 72 h by standard MTT assay.
B, time course of staurosporine-induced apoptosis. Cells
were treated with 10 ng/ml staurosporine, incubated with 7AAD, and
analyzed for apoptosis by flow cytometry. C, morphological
analysis of A375 and UACC 1227 cells treated with staurosporine (10 ng/ml) for 72 h. Magnification ×100. a, A375
nontreated cells; b, A375 cells treated with staurosporine;
c, UACC 1227 nontreated cells; d, UACC 1227 cells
treated with staurosporine.
Blockage of Fas-induced cell death by caspase 3 and caspase 8 inhibitors
View larger version (39K):
[in a new window]
Fig. 7.
Stimulation of PITSLRE kinase activity during
anti-Fas mAb- or staurosporine-induced apoptosis is partially blocked
by caspases 3 and 8 inhibitors in A375 cells. Histone H1 was used
as the substrate for the assays. Autoradiograph of histone H1 kinase
activity at 48 h following treatment with anti-Fas mAb,
staurosporine, and/or caspase 8 and caspase 8 inhibitors. Relative
kinase activity from the phosphorylated histone H1 band was determined
by phosphoimaging.
and isoforms
(Fig. 8, A and B).
Conversely, in UACC 1227 cells, p110 PITSLRE isoforms have a
cytoplasmic localization (Fig. 8C). These results clearly
demonstrate that the localization of p110 isoforms in UACC 1227 cells
is different from that of normal melanocytes. In addition, because UACC
1227 cells are resistant to Fas-mediated apoptosis, nuclear
localization of p110 isoforms may be necessary for the complete
activation of the Fas signaling pathway.
View larger version (31K):
[in a new window]
Fig. 8.
Localization of p110 PITSLRE protein kinase
in normal melanocytes and cancer cells by immunofluorescence confocal
microscopy. Comparison of p110 PITSLRE kinase isoforms
localization using the affinity-purified P2N100 polyclonal antibody and
double immunofluorescence staining in normal melanocytes
(A), A375 cells (B), and UACC 1227 cells
(C). PITSLRE p110 isoforms localize to the nucleus in normal
melanocytes and A375 cells. Conversely, in UACC 1227 cells, the PITSLRE
p110 isoforms have a cytoplasmic localization.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (29K):
[in a new window]
Fig. 9.
PITSLRE model of cell death pathways in
melanoma. Three signals for cell death are illustrated: 1)
staurosporine, 2) Fas/Apo-1, and 3) DR3.
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed: Arizona Cancer
Center, Rm. 3963 B, 1515 N. Campbell Ave., Tucson, AZ 85724. Tel.: 520-626-4515; E-mail: mnelson@azcc.arizona.edu.
ABBREVIATIONS
-D-galactopyranoside;
RT, room temperature;
PARP, poly ADP-ribose polymerase.
REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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