Early-phase events of genotoxin-induced apoptosis have been shown to involve activation of JNK1. 1,2 In contrast, the MEK1/ERK pathway, whose role in proliferative signalling is well known, has been suggested to have an anti-apoptotic role, in that the balance between the ERK and JNK pathways may be important in determining whether a cell survives or undergoes apoptosis. 3,4 Activation of MEK1/ERK can also protect against Fas-induced apoptosis;5 similarly, phorbol ester-mediated inhibition of drug-induced apoptosis was shown to involve activation of ERK. 6 Furthermore, inhibition of the ERK pathway may on its own induce apoptosis, 7 or may enhance apoptosis induced by other agents. 4
Cisplatin is widely used as a DNA-damaging anticancer drug and is known to induce apoptosis in a range of different tumour cell lines. 8 Accordingly, cisplatin has been shown to lead to activation of JNK1 and caspase-3. 9,10 Cellular stress and DNA damage, such as that induced by cisplatin, lead to activation of specific genes; however, the Pt-DNA adduct levels in cisplatin-treated cells do not correlate with gene activation levels. 11 Thus, in addition to actual adduct formation and DNA damage, downstream signalling pathways and their relative levels of activation may also be of importance for the outcome of cisplatin treatment.
The MEK1 inhibitor PD98059 has recently been shown to sensitize two ovarian cancer cell lines to cisplatin. 12 Because cisplatin resistance is a serious clinical problem, and because of the suggested role of the MEK1/ERK pathway in anti-apoptotic signalling, we used a panel of cisplatin-resistant human melanoma cell lines to study the effects of PD98059 on the cisplatin responses of these cell lines.
Materials and methods
All the cell lines were maintained in RPMI medium supplemented with 10% fetal calf serum (both from Life Technologies), l-glutamate, penicillin and streptomycin, and were grown at 37°C in 5% CO2.
Cells were seeded (1 million/dish) and after 24 h treated with various doses of cisplatin. After various periods of incubation with cisplatin, the cells were rinsed and harvested for the respective analyses.
Cells were seeded in triplicate at a density of 5000/well in 96-well plates and were treated 30 h later with various doses of cisplatin for 24 h. PD98059 40 μM (New England Biolabs, Inc.) was added to cells either 20 h before the cisplatin, or concomitantly with it. Viability after treatments was detected using an MTT assay (Cell Titer 96 Assay, Promega) according to the manufacturer's instructions. The resulting absorbance measured at 595 nm is directly proportional to the number of viable cells. Viability is shown as a percentage of the viability in an untreated control.
Extracts of cells treated for 4 h with 40 μM cisplatin were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE); 20 μg protein/sample was transferred onto PVDF membranes and probed with an antibody that primarily recognizes phosphorylated JNK1 and JNK2, but also crossreacts with phosphorylated ERK1 and ERK2 (New England Biolabs, Inc.). The identity of the ERK bands was confirmed with ERK-specific antibodies (Santa Cruz Biotechnology, Inc.). Bound antibodies were detected using secondary horseradish peroxidase (HRP) conjugated antibodies and ECL chemoluminescence reagents (Amersham).
Detection of apoptosis
Cells were treated with cisplatin as indicated, rinsed in phosphate buffered saline (PBS) and harvested using Cell Dissociation Solution (Sigma Aldrich AB, Sweden). The cells were then suspended in a small volume of hypotonic salt solution with 30 mM glycerol. Smears were made on glass slides and, after airdrying, the cells were fixed in acetone-methanol (2:1) for 5 min. DNA was visualized by immersing the slides in a solution of ethidium bromide in distilled water (5 ng/ml) for 5 min. After rinsing in tap water, cells stained with ethidium bromide were examined by ultraviolet (UV) microscopy. The percentage of cells with fragmented DNA in each sample was assessed. Approximately 100 cells per sample were counted. Apoptosis was also assessed by terminal deoxynucleotidyl transferase-mediated. dUTP nick end labelling-fluorescein isothiocyanate (TUNEL-FITC) fluorescence activated cell sorter (FACS) analysis, using the In Situ Cell Death Detection kit (Boehringer Mannheim, Roche Molecular Biochemicals) according to manufacturer's instructions, and co-analysed with DAPI staining by flow cytometry as described below.
C8161, AA and DFW cells were pretreated with 40 μM PD98059 or vehicle for 20 h before the addition of cisplatin (40 μM). After 24 h, cell extracts were made for caspase-3 activity assays using the CaspACE system (Promega Corporation), according to the manufacturer's instructions.
Control and cisplatin-treated cells were harvested with cell dissociation solution (Sigma Chemical Co.), pelleted and resuspended in 4 ml of fresh buffered 4% formaldehyde solution. After fixation for 30 min at room temperature, the cells were pelleted, resuspended in dropwise added ice-cold 95% ethanol during vortexing, and stored at −20°C. Flow cytometric analysis was performed using a Partec Flow Cytometer PASII (Partec, Munster, Germany). Aliquots of the cell material fixed with formaldehyde and stored in ethanol were washed once in tap water. The cells were transferred to 0.2 ml subtilisin Carlsberg solution (0.1% Sigma protease XXIV, 0.1 M Tris, 0.07 M NaCl, pH 7.2) and incubated for 45 min at 30°C to release the cell nuclei. These were stained by the direct addition of DAPI solution. At least 15,000 cell nuclei were analysed per sample. The cell cycle composition was calculated using the MulticycleAV program (Phoenix Flow Systems, San Diego, California, USA).
Sensitivity of human melanoma cells to cisplatin
A panel of six melanoma cell lines was exposed to different concentrations of cisplatin. Apoptosis was assayed 24 h later by scoring nuclear fragmentation using ethidium bromide staining (Table 1). Two cell lines were highly sensitive to cisplatin (FM55 and 224) and four cell lines (AA, DFW, C8161 and M5) were found to be more resistant and were studied further.
Effect of PD98059 on melanoma cell growth
PD98059 is an inhibitor of MEK1 (MAP kinase-ERK kinase). The effect of PD98059 on the cell growth of AA, DFW, C8161 and M5 cells was examined using the MTT assay. PD98059 treatment retarded the cell growth of all four resistant cell lines, although the C8161 and AA cell lines were more affected than DFW and M5 (Figure 1).
Basal and cisplatin-induced phosphorylation of ERK1–2 and JNK1–2
The basal phosphorylation levels of JNK2 and ERK2 were very low in the DFW, C8161 and AA cell lines, while basal ERK1 phosphorylation was noticeably higher in C8161 cells than in the other two lines (Figure 2). Basal levels of phosphorylation of these kinases were very low in M5 cells (data not shown), in accordance with earlier reports. 13,14 After 4 h of cisplatin treatment (40 μM), the DFW, C8161 and AA cell lines showed various degrees of increased phosphorylation of all four kinases (Figure 2a). JNK2 activation was prominent in DFW and AA cells, but less so in C8161. The fold increase in JNK1 phosphorylation from the basal level was approximately similar in all three lines, in accordance with similar levels of cisplatin sensitivity. Similarly, induction of ERK1 phosphorylation was approximately equal in these three cell lines. In contrast, ERK2 induction was more prominent in C8161 cells than in the other two cell lines. No or very weak increases in ERK and JNK were observed in M5 cells (data not shown), in accordance with previous observations. 13,14 PD98059-mediated inhibition of basal and cisplatin-induced ERK phosphorylation was also confirmed in C8161 cells (Figure 2b).
PD98059 sensitizes C8161 cells to cisplatin
The effect of PD98059 on cisplatin-induced cytotoxicity was examined using the MTT assay (Figure 3). Cells were pretreated for 20 h with PD98059 before the addition of cisplatin, and viability was assayed 24 h later. PD98059 was found to have no effect on the cisplatin sensitivity of DFW and M5 cells, while a slight protective effect on AA cells was observed. In contrast, pretreatment with PD98059 had a significant sensitizing effect on C8161 cells (Figure 3d). This sensitization required pretreatment with PD98059, since concomitant addition of PD98059 and cisplatin did not result in any increase in cytotoxicity (Figure 3e).
Analysis of the sensitization of the cisplatin response in C8161 cells was repeated using TUNEL staining (Figure 4). Pretreatment with 40 μM PD98059 followed by 40 μM cisplatin for 24 h in the presence of PD98059 induced extensive apoptosis in C8161 cells, whereas either agent alone induced little or no apoptosis at these concentrations.
Treatment of C8161, DFW and AA cells with 40 μM cisplatin for 24 h led to the activation of caspase-3 (Figure 5). In cells pretreated with 40 μM PD98059 there was no further increase in caspase-3 activity; on the contrary, there was a minor decrease (Figure 5).
Cell cycle phase distribution
The effects of PD98059 and cisplatin on the cell cycle phase distribution of C8161 cells was analysed (Table 2). Treatment with PD98059 for 44 h, i.e. the total time for the sensitization experiments, resulted in a decreased S-phase fraction and an increase in the G2 fraction. Together this indicates a G1 and G2 block. Cisplatin was found to cause S-phase arrest at all the concentrations tested, although the block was especially prominent with 20 μM cisplatin. Preincubation with PD98059 for 20 h did not greatly alter the cell cycle phase distribution induced by 40 μM cisplatin.
Although many cellular mechanisms of resistance to cisplatin have been defined, such as those mediated by the glutathione/glutathione S-transferase system and increased DNA repair systems, 15 the apoptotic signalling induced by cisplatin in tumour cells has only been partly characterized, and cisplatin resistance mechanisms relating to these signals are even less well understood. 15 It is known, however, that activation of the SAPK/JNK pathway is involved in cisplatin-induced apoptosis. 2,9,16 The role(s) of the MEK1/ERK pathway are unclear; however, it was recently shown that PD98059, which inhibits MEK1, the only known activating kinase directly upstream of ERK, sensitized two ovarian carcinoma cell lines to cisplatin-induced cell death. 12 We have here investigated whether cisplatin-induced cell death in human melanoma cell lines can be similarly potentiated.
In accordance with earlier reports, 2,9,16 we found that JNK1 was activated by 40 μM cisplatin in three out of the four cell lines tested. It has been suggested that activation of JNK2 may be part of a protective response rather than a pro-apoptotic one. 17 We report here that two of the relatively resistant melanoma cell lines showed a prominent JNK2 activation by cisplatin – a lack of correlation that probably reflects the existence of several parallel mechanisms of resistance.
In three of the melanoma cell lines studied here, basal ERK phosphorylation levels were low compared with those in C8161 cells, in which especially ERK1 was highly phosphorylated. Induction of ERK1 phosphorylation by cisplatin was similar in DFW, C8161 and AA cells, while in C8161 cells it induced a particularly pronounced ERK2 phosphorylation. There was thus no obvious correlation between cisplatin-induced apoptosis and ERK phosphorylation in these cell lines.
In order to study the role of ERK in the cisplatin response, the effects of PD98059 in combination with cisplatin were studied using the MTT assay. The cisplatin responses of DFW and M5 cells were not affected by preincubation with PD98059, whereas a slight protective effect was observed in AA cells. Interestingly, C8161 cells showed a substantial loss of viability when pretreated with PD98059 before cisplatin treatment. This sensitizing effect was confirmed using TUNEL-FACS, where massive DNA fragmentation was seen in similarly treated C8161 cells. This additional fragmentation does not appear to be mediated by caspase-3, since PD98059 pretreatment did not increase cisplatin-induced caspase-3 activity. Instead, PD98059 pretreatment might lead to an increase in an unidentified non-DEVDase activity shown to be involved in oligonucleosome-size DNA fragmentation, while caspase-3 activity is responsible for high molecular weight DNA fragmentation. 18 Non-caspase proteases have also been shown to alter chromatin structure, thereby facilitating apoptosis-related DNA fragmentation. 19
It appears that upstream of DNA fragmentation, the actual sensitizing effect of PD98059 on the cisplatin response of C8161 cells involves downregulation of a cell-specific, ERK1-mediated signal. This is based on the requirement for preincubation with PD98059 in order to achieve potentiation, and on the observation that the basal activity of ERK1 is significantly higher in C8161 cells than in DFW and AA cells. Since the inhibitory effect of PD98059 on MEK1 activation is rapid, i.e. it occurs within minutes, 20 the preincubation requirement suggests that the sensitization mechanism involves several steps, such as the downregulation of viability-promoting proteins regulated by ERK1.
We furthermore suggest that the protective effect of PD98059 on the cisplatin response of AA cells is due to the growth retardation induced by PD98059 in these cells. PD98059 also induced significant growth retardation in C8161, probably by causing blocks in G1 and G2 (Table 2), but this effect was overridden by the loss of a key viability-promoting signal, as argued above.
In summary, although many reports have shown a role for the MEK1/ERK pathway in viability-promoting signalling, our data show that inhibition of this pathway is not necessarily a general tool for sensitizing human cancer cells to cisplatin. They also illustrate the complexity of cellular signalling, in that the outcome of a given treatment depends on balances between the activities of several different, often cell-specific pathways.
This work was supported by grants from the Swedish Cancer Society and the Gustaf V Jubilee Foundation and the Foundation for Strategic Research (Sweden).
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