Fas-induced Apoptosis in Human Malignant Melanoma Cell Lines Is Associated with the Activation of the p34 cdc2 -related PITSLRE Protein Kinases*

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␤-converting enzyme (ICE-like) family, which are currently considered to be the central executioners of apoptosis (13)(14)(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.
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 p34 cdc2related kinases whose functions appear to be linked to control of cell division and possibly programmed cell death (22)(23)(24)(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␣-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
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 g/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 g/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 NaN 3 , 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 g/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 g) and the melanoma cell lines A375, UACC 1227, and UACC 903 was analyzed for distinct mRNA species using PharMingen's RiboQuant TM multi-probe with the hAPO-2, hAPO-3, and hAPO-5 probe template sets. ␣-32 P-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 (Ϫ80°C, overnight).
Cell Treatment-On day 1, A375 and UACC 1227 cells (1 ϫ 10 6 ) 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% CO 2 environment. On day 0, cells were washed twice with PBS and treated with either 0.5 g/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 ϫ 10 6 ) were stained with 7AAD (200 g/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 7AADnegative) was validated by cell sorting and morphological examination.
Cell Morphology-Cytospin preparations of anti-Fas mAb and staurosporine-treated and nontreated control cells (1 ϫ 10 5 ) 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 airdried and analyzed by light microscopy at 100ϫ.
MTT Assay-A375 and UACC 1227 cell viability was assessed using the MTT assay. Cells (2 ϫ 10 4 /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 g/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% Me 2 SO 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.
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 ␤-actin(1:8000) antibodies as described previously (21,31). Briefly, protein extracts (either 30 or 50 g) from control and treated samples were separated by SDSpolyacrylamide 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).
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/l in 50 mM Hepes, 100 mM KCL, 40 mM Na 2 PO 4 , 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 ␤-galactosidase activity. After an overnight incubation, the blue cells in each field were quantitated by visual inspection. For the transfection experiments, 5 ϫ 10 5 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-galstained cells in 20 random fields were quantitated for each dish.
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 Ϫ20°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 Cy5streptavidin for 1 h, washed three times with PBS, and incubated with RNase (100 g/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.
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 Fasinduced 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 mAbmediated 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 ϫ 10 6 ) 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 ␤-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.
A375 and UACC 1227 Cells Differ in their Sensitivity to Staurosporine-induced Apoptosis-To determine whether alternative apoptotic pathways exist in UACC 1227 cells, stau-rosporine 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.  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 fol-

lowing 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-Fasand 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 trans-formed 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 ␣ 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 Fasmediated apoptosis, nuclear localization of p110 isoforms may be necessary for the complete activation of the Fas signaling pathway. DISCUSSION 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 aminoterminal 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  a A375 cells were treated as described under "Materials and Methods," and cell survival was determined using the standard MTT assay.

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.
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.