Department of Microbiology and Immunology, C4-101 VMC, Cornell University, Ithaca, New York 14853
Received January 11, 2002; accepted March 7, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: sodium arsenite; Burkitt's lymphoma; MAP kinase; JNK; p38; apoptosis; hyperthermia.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three major MAP kinase pathways, designated by their terminal kinases, have been extensively studied: the extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK1/2), and p38 kinase pathways. These MAP kinases are activated via a series of sequential phosphorylations of upstream kinases, and they function primarily to transduce signals to the cell nucleus, ultimately affecting gene expression. Among the targets of the MAP kinase pathways induced by arsenite (Liu et al., 2001) are components of the activator protein 1 (AP-1) transcription factor, a heterodimer or homodimer consisting of Fos/Jun or Jun/Jun proteins that regulate the expression of multiple genes, including those encoding the Fos and Jun proteins. Generally, activation of the ERK pathway is associated with proliferation, whereas activation of the JNK and p38 pathways is associated with the induction of apoptosis by various physical and chemical stresses. However, it is now apparent that the role of each MAP kinase pathway is determined by multiple factors, including cell type, the particular isoform of the activated kinases, the duration of kinase activation, and other regulatory signals.
Several published studies demonstrate that the complex dose-dependent and cell type-specific outcomes of arsenic exposure involve the differential activation of MAP kinase pathways. For example, activation of the ERK pathway by low concentrations of sodium arsenite-induced transformation of mouse epidermal cells in vitro, whereas exposure to higher arsenite concentrations (>50 µM) resulted in JNK pathway activation and apoptosis induction (Huang et al., 1999a,b
). Chronic, low-level (<6 µM) arsenite exposure of C3H 10T1/2 fibroblasts, a model system of in vitro carcinogenesis, disrupted the normal regulation of proliferative signals mediated via the receptor tyrosine kinase pathway. This pathway is an upstream activator of ERK and is believed to be responsible for the mitogenic effect of arsenite in this system (Trouba et al., 2000
). Similarly, acute exposure of vascular endothelial cells to low levels of arsenite (15 µM) elicited a mitogenic response, which correlated with the generation of oxidative stress and the activation of tyrosine-kinase signaling cascades, but not with stress-kinase activation. In contrast, this same study revealed that the activation of p38 kinase required high concentrations of arsenite (100 µM) and was associated with cell death (Barchowsky et al., 1999
). Together, these and other published studies (Ludwig et al., 1998
) clearly attribute the ability of arsenite to induce apoptotic cell death to activation of the JNK and/or p38 pathways. Invariably, however, high concentrations of arsenite, often in excess of 100 µM, were required to elicit an apoptotic response in those experimental systems.
Understanding the cellular and molecular basis of arsenite-induced alterations in cells requires the systematic analysis of key regulatory pathways that may be involved, as well as the innate characteristics of the cell type that may confer stress resistance or sensitivity. In addition, as suggested by the above studies, mechanisms of arsenite-induced cytotoxic effects need to be understood in the context of cell type and arsenic concentration, especially at environmentally relevant levels. B-lymphoid cells are particularly attractive for such studies, as this cell type and various established cell lines are highly sensitive to several drugs and environmental toxicants (Smith and Sladek, 1985; Wilmer et al., 1992
). Burkitt's lymphoma (BL) cell lines have proven to be a particularly useful model system of human germinal center-derived B-lymphocytes. They have been important in studies on the induction of apoptosis by engagement of the surface IgM receptor and the subsequent rescue from IgM-induced apoptosis by CD40, critical components of negative/positive selection of B-lymphocytes during development (Hornung et al., 1998
; Kaptein et al., 1996
; Sakata et al., 1995
, 1999
). Importantly, although these studies invoke the differential activation of various MAP kinase pathways in receptor-mediated processes, little is known concerning the signaling pathways that regulate the induction of apoptosis by chemical agents in these cells.
Similar to B-lymphocyte subpopulations in vivo, individual BL cell lines show differences in their sensitivity to apoptosis induction after exposure to radiation and a variety of drugs and toxic chemicals (Fan et al., 1994; Gaidano et al., 1991
; Lin et al., 1998
; O'Brien et al., 2001
). This differential sensitivity cannot be attributed solely to the cellular factors commonly associated with regulation of apoptosis, such as p53 status, drug efflux, or glutathione level (Fan et al., 1994
; Gaidano et al., 1991
; Lee and Shacter, 1997
; Merino et al., 1994
; Uckun et al., 1991
). Thus, other cellular systems are likely responsible for regulating sensitivity to chemically induced apoptosis in BL cells.
We recently validated a panel of BL cell lines that demonstrate significant differences in their susceptibility to apoptosis induction as a model system to examine the association of MAP kinases with chemically induced apoptosis (O'Brien et al., 2001). The ST486 line shows sensitivity to anticancer drugs and mitochondrial toxicants and has a high ratio of Bax/Bcl-2, proapoptotic, and antiapoptotic members, respectively, of the Bcl-2 family of proteins. In contrast, the EW36 cell line shows multidrug resistance and has a low Bax/Bcl-2 ratio because of the overexpression of the antiapoptotic Bcl-2 protein. Both cell lines have similar drug efflux characteristics and cell cycle transit profiles.
The present study was undertaken to compare the sensitivity of these cell lines with sodium arsenite over a wide range of concentrations and to investigate the role of MAP kinase signaling pathways in regulating chemically induced apoptosis. We specifically addressed the requirement for activation of the JNK pathway in apoptosis induction in the ST486 compared with EW36 cell lines. In addition, we studied the interaction of other stresses, specifically nonlethal heat shock, with low-dose arsenite exposure in EW36 cells to determine whether synergistic interactions of sublethal treatments can sensitize resistant cells to apoptosis induction (and whether such interactions are mediated by MAP kinase activation).
Importantly, we found a significant difference in the requirement for JNK pathway activation in arsenite-induced apoptosis between the two cell lines. Whereas the EW36 cells underwent apoptosis at relatively high arsenite concentrations in a manner associated with JNK pathway activation, ST486 cells underwent apoptosis at low-to-moderate concentrations in a JNK-independent manner. Moreover, sublethal hyperthermia, which itself induced only a transient activation of JNK in EW36, acted synergistically by lowering the threshold concentration of arsenite required to activate the JNK pathway and to induce apoptosis. We also found that thermal sensitization of EW36 cells can be further enhanced by inhibition of the p38 kinase pathway. In all cases, sensitization of the resistant line involved the sustained activation of the JNK pathway and was mediated, at least in part, by activation of its upstream kinase, SEK1.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell lines and culture conditions and determination of growth inhibition.
The BL cell line ST486 was obtained from ATCC, Rockville, MD. The EW36 cell line was obtained from Dr. Ian T. Magrath, National Cancer Institute, Bethesda, Maryland. Both of the cell lines grow optimally and similarly in the same medium formulation, have doubling times of approximately 24 h, and are Epstein-Barr virus-negative. The cell lines were cultured in medium RPMI 1640 (GIBCO) supplemented with 15% fetal calf serum, penicillin-streptomycin, and L-glutamine. Cells were grown at 37°C, 5% CO2, and 95% humidity.
For all experiments, cultures were set up at a density of 0.4 x 106 cells/ml and allowed to grow for 24 h. After appropriate pretreatment (i.e., +/ heat shock at 43°C for 1 h or +/ SKF), cells were plated into six well plates at 3 ml/well, and sodium arsenite was added. At the designated times, cells were harvested for protein immunoblotting or for detection of morphological apoptosis as described below. In addition, cell counts were performed at 12, 24, 36, and 48 h after chemical addition, using a Coulter model ZM counter and channelyzer. Increases in cell number over time were determined for a series of graded doses of arsenite ranging from 1 to 50 µM. ID50s were derived based on a comparison of the extent of growth inhibition for each concentration of arsenite compared with untreated control.
Cytological detection of apoptosis and necrosis with the H/PI assay.
The induction of apoptosis was analyzed using a double fluorescence staining technique (Muscarella and Bloom, 1997; Muscarella et al., 1998
). The procedure allows simultaneous detection of plasma membrane integrity by dye exclusion and apoptotic phenotypes by observing condensed, segregated chromatin in live cells. Briefly, cells were stained in 20 µg/ml propidium iodide (emitting red fluorescence) and 100 µg/ml Hoechst 33342 (emitting blue fluorescence) for 15 min at 37°C in the dark. The double fluorescence was detected with a Leitz Aristoplan microscope equipped with an epifluorescence system and a long-pass filter cube A. Dead cells emit red and live cells emit blue fluorescence. Apoptotic cells have a characteristic phenotype of condensed, segregated chromatin in intact but shrunken cells (fluorescing blue in early stages and red later on). The apoptotic phenotype was easy to detect and discriminate from necrotic cells, which were swollen, had irregular/damaged membranes, and were PI-positive. The chromatin was minimally condensed, with some accumulation near the nuclear membrane. Typically, 200 cells were scored for each sample and were classified as either necrotic, apoptotic, or normal/viable.
Statistical analysis of the data.
Statistical analysis of apoptosis induction was as follows: 200 cells were scored per sample and data were entered into a database for subsequent analysis. Percentages (e.g., percent apoptosis) were transformed by arc sin to normalize the data. A one-way analysis of variance (ANOVA) was performed on data sets from experiments including control and treatment values. If the F-statistic was significant, post hoc comparisons were made using Fisher's LSD test.
Protein immunoblotting.
After chemical exposure for the specified period of time, cells (1 ml of culture) were collected, washed in phosphate buffered saline (PBS), and solubilized in 50 µl of 1 x Laemmli sample buffer (65.2 mM Tris-Cl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromphenol blue, and 5% ß-mercaptoethanol). Ten microliters of lysate (approximately 4 x 105 cells/sample) were subjected to SDS-PAGE in a 415% gradient gel. Gels were electrophoretically transferred to nitrocellulose membrane (Bio-Rad) in 25 mM Tris, pH 8.3, 192 mM glycine, and 20% MeOH. For detection of phosphorylated kinases, membranes were first probed with antibodies specific for the phosphorylated forms of ERK1/2, JNK1/2, and p38 (New England Biolabs). Filters were subsequently reprobed using antibodies that recognize the proteins independent of phosphorylation status to ensure that differences in signal were due to phosphorylation of the protein and not to differences in amounts of total protein. For PARP cleavage, an antibody that recognizes the 113-kD, intact PARP, and the 85-kD cleavage product was used to simultaneously detect cleaved and uncleaved PARP (Stressgen, Inc.). Membranes were washed in TBS (20 mM Tris, 500 mM NaCl, pH 7.5), then blocked for 1 h in TBS containing 5% dried milk. Filters were washed in TBS containing 0.1% Tween-20 and incubated overnight at 4°C with primary antibody diluted appropriately in TBS containing 5% bovine serum albumin. Filters were washed again and incubated with the second antibody, horseradish peroxidase conjugate. Detection was then performed using an enhanced chemiluminescent (ECL) system. Quantitation of the signals was performed using an Alpha Imager 2000 Documentation and Analysis System, equipped with AlphaEase version 3.2 software (Alpha Innotech Corp.).
MAP kinase activity assay.
Kinase activity assays were performed using a commercially available kit (Cell Signaling, Inc.). Briefly, 2 h after chemical exposure, cells were lysed on ice in a buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Lysates were sonicated and p38 was immunoprecipitated by gentle agitation overnight at 4°C using a monoclonal antibody for phosphorylated p38 immobilized to sepharose beads. Antibody complex was collected by centrifugation and, after washing, was incubated for 30 min at 30°C in kinase buffer containing 25 mM Tris (pH 7.5), 5 mM ß-glycerolphosphate, 2 mm DTT, 0.1 mM Na3VO4, and 10 mM MgCl2, plus 2 mg/ml of a GST-ATF2 fusion protein as the substrate. Phosphorylation of the substrate was detected by subjecting the kinase reaction to electrophoresis and immunoblotting as described above, using an antibody specific for the phosphorylated form of ATF2.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In contrast to apoptosis induction, we found that growth inhibition by arsenite was similar for the two lines, with ID50 values of 5 µM and 7 µM for ST486 and EW36 cells, respectively. Complete growth arrest occurred at a concentration of 20 µM for both cell lines. Although these data do not prove similar uptake of arsenite in these cell lines, they suggest this is the case, and indicate that growth arrest and apoptosis induction by arsenite are separable processes. Moreover, these findings are consistent with our previous findings of similar P-glycoprotein pump activities in these lines (O'Brien et al., 2001) and provide the basis for identifying differences between sensitive and resistant BL cell lines in arsenite-induced signaling for apoptosis.
BL Cell Lines Show Differences in the Association of JNK Pathway Activation with Arsenite-Induced Apoptosis
Activation of the JNK pathway is associated with arsenite-induced apoptosis in a number of cell types. We determined the extent of activation of this kinase pathway in the BL cell lines and its association with arsenite-induced apoptosis. Activation of this pathway was assessed in two ways. First, phosphorylated JNK was identified in cell lysates 2 h after arsenite exposure by protein immunoblotting using antibodies specific for the dual-phosphorylated, and by inference active, forms of the two SAPK/JNK isoforms, JNK1 and JNK2 (Fig. 2A; p-JNK1 and p-JNK2). Filters were then reprobed with an antibody that detects each kinase regardless of its phosphorylation state (Fig. 2A
; JNK1 and JNK2) to ensure equal loading of each lane and to serve as an internal control for subsequent quantitation. Second, the induction of one of the downstream targets of the JNK pathway, c-Jun, was determined 24 h after arsenite exposure, at the same time as PARP cleavage. The data show that the ST486 cells underwent extensive apoptosis/PARP cleavage at arsenite concentrations below those required for activation of the JNK/c-Jun pathway. In contrast, EW36 cells showed PARP cleavage only at concentrations of arsenite that activated this pathway, with neither apoptosis induction nor JNK pathway activation detected at lower arsenite concentrations.
|
For both cell lines, phospho-JNK1 and 2 were detected 1 h after arsenite addition and sustained for several hours following exposure (Fig. 2B; p-JNK1 and p-JNK2). There was some reduction in the signal for phopsho-JNK2 in ST486 at 8 h and for phospho-JNK1 and JNK2 in EW36 cells at 18 h. As this loss of phospho-JNK signal coincided with the induction of apoptosis, we are unable to attribute it to specific differences in the regulation of phospho-JNK between the cell lines. Overall, however, the two cell lines showed relatively similar profiles of JNK phosphorylation during the first several hours of arsenite exposure.
In contrast, the cell lines differed significantly with respect to the kinetics of c-Jun induction and PARP cleavage. EW36 cells showed a clear time-related increase in c-Jun protein that preceded the appearance of cleaved PARP. Moreover, no morphological apoptosis was detected in EW36 cells during the first 8 h of exposure. In contrast, ST486 cells showed extensive PARP cleavage at 4 h, which occurred in the absence of detectable c-Jun. Levels of morphological apoptosis detected by the fluorescence assay paralleled the rapid induction of PARP cleavage: 50% of ST486 cells showed morphological apoptosis at 6 h. However, c-Jun protein was minimally induced during this period. Thus, we surmise that early and extensive apoptosis in the ST486 cultures occurs before they can accumulate measurable levels of c-Jun protein. These data suggest that, whereas early signaling for phospho-JNK by arsenite is similar between the cell lines, apoptosis induction in ST486 cells is rapid and involves signals independent of the induction of downstream products of the JNK/c-Jun pathway. In contrast, EW36 cells show clear temporal and concentration-dependent associations of JNK phosphorylation, c-Jun induction, and apoptosis.
Nonlethal Hyperthermia Sensitizes EW36 Cells to Arsenite-Induced Apoptosis
EW36 cells overexpress Bcl-2 (Lee and Schacter, 1997; O'Brien et al., 2001), with approximately 50-fold higher levels of this protein compared with ST486 and other BL cell lines. Our observed association of the JNK/c-Jun activation/induction with arsenite-induced apoptosis suggests that manipulation of this pathway may be effective for bypassing Bcl-2mediated resistance to apoptosis induction in EW36 cells. Hyperthermia activates the JNK pathway and induces apoptosis in lymphoma and leukemic cell lines (Buzzard et al., 1998
; Katschinski et al., 1999
). Therefore, we were interested in whether the BL cell lines are differentially sensitive or resistant to treatment with hyperthermia and whether exposure to heat shock may be used as an alternative way of activating the JNK pathway in resistant cells.
We found that ST486 cells underwent rapid and extensive apoptosis after exposure to heat shock of 43°C for 60 min, approaching 100% morphological apoptosis (data not shown) and PARP cleavage at 24 h (Fig. 3A). In contrast, the same heat-shock treatment was not lethal to EW36 cells. However, the EW36 cell line showed substantial sensitization toward a previously sublethal exposure to arsenite when first subjected to this nonlethal heat-shock treatment (Figs. 3B and 3C
). The number of cells exhibiting morphological apoptosis increased from an average of approximately 25% in arsenite-treated cultures to 70% in cultures exposed to heat shock plus arsenite (Fig. 3C
). The extent of PARP cleavage increased in a similar manner (Fig. 3B
; PARP).
|
Because we observed enhanced activation of both the p38 and JNK pathways in EW36 cells sensitized by hyperthermia, we performed experiments to further understand the potential contribution of these two pathways in the acquired sensitivity of this cell line. For these experiments we used several pharmacological inhibitors of p38 kinase: SB202190 (SB2), its analogues, SKF86002 (SKF) and SC68376, or an inactive analogue, SB202474 (SB-inact). We found that treatment of cell cultures with each of the active p38 inhibitors gave similar results. However, SB2 on its own induced ERK phosphorylation in the BL cell lines, whereas SKF did not. The ability of some p38 inhibitors to activate the ERK pathway via Ras/Raf signaling has been reported (Hall-Jackson et al., 1999). However, to avoid the potential confounding effects of ERK activation, we used SKF in the experiments reported here.
The Hsp27 stress protein is induced by arsenite and phosphorylated by MAPKAP kinase2, which is a substrate of p38 kinase. Using an antibody specific for phospho-Hsp27 (p-Hsp27) or total Hsp27, we determined the concentration of SKF required to substantially inhibit p38-mediated Hsp27 phosphorylation in EW36 cells following arsenite exposure (Fig. 4A). SKF gave a concentration-dependent decrease in phosphorylation of Hsp27 without affecting the overall level of induction of the protein. For the experiments described, SKF at a concentration of 20 µM was used, as substantial inhibition of Hsp27 phosphorylation (70%) was achieved at this level, and it is below the concentrations for which nonspecific effects of the p38 inhibitors have been reported.
|
These data suggest that heat shock and p38 inhibition both contribute to the sensitization of EW36 cells. Furthermore, these experiments suggest that sensitization by the p38 inhibitiors is not the result of nonspecific activity of the compounds. First, sensitization of EW36 cells occurred at the same SKF concentration required to substantially inhibit p38 activity, as measured by the reduction in phosphorylated Hsp27. Second, pretreatment of cells with the structural analogue that is unable to inhibit p38 (Young et al., 1997), confirmed in our study by its inability to block phosphorylation of Hsp27 (data not shown), failed to sensitize EW36 cells to arsenite-induced apoptosis. The profiles of PARP cleavage and c-Jun induction were similar for cultures pretreated with the inactive analogue as for cultures exposed to arsenite alone (Fig. 4B
; compare As with SB-inact + As). It is interesting to note that SKF treatment did not similarly sensitize the ST486 cells to apoptosis induction by arsenite (data not shown). However, this is consistent with our previous experiments that indicate arsenite-induced apoptosis occurs rapidly, in a manner independent of activation/induction of the JNK/c-Jun pathway, in the sensitive cell line.
The above experiments suggest that the JNK and p38 pathways have opposing effects on arsenite-induced apoptosis in the EW36 cell line, with inhibition of p38 sensitizing cells to apoptosis induction in a manner associated with activation of the JNK/c-Jun pathway. To further explore the requirement of the JNK/c-Jun pathway in apoptosis induction, cultures of EW36 cells were pretreated with oleandrin. This bioflavanoid inhibits the AP-1 transcription factor (Manna et al., 2000), a downstream target of the JNK pathway that activates expression of the c-jun gene. Consistent with published data, we found that treatment of EW36 cells with oleandrin inhibited the induction of c-Jun by arsenite (Fig. 4C
). Importantly, oleandrin also abolished PARP cleavage in these cultures. However, it did not block the induction of phospho-JNK by arsenite (data not shown). Together, these data suggest that targets downstream of JNK and associated with AP-1 activation are required for apoptosis induction/PARP cleavage in the EW36 cells.
Nonlethal Hyperthermia Induces Transient Phosphorylation of JNK in EW36 Cells That Is Prolonged by Inhibition of p38
To understand the potential interaction of p38 inhibition and hyperthermia with induction of JNK/c-Jun, we analyzed the kinetics of kinase activation during a time course consisting of varying lengths of exposure to heat shock with and without a period of recovery (Fig. 5A). Cultures of ST486 or EW36 cells were subjected to heat shock for 30, 60, or 90 min. Samples were taken either immediately after heat shock (t0) or after a 4-h recovery at 37°C (t4) and analyzed for presence of phosphorylated JNK and p38. We found that phospho-JNK1/2 was induced after heat shock for both cell lines, but its phosphorylation was transient in EW36 cells, resulting in a substantially reduced signal at 4 h. This transient phosphorylation of JNK in EW36 is consistent with the results shown in Fig. 3B
. In that experiment, arsenite was added to cultures 4 h post-heat shock and cultures were analyzed 2 h later, for a total of 6 h post-heat shock. Those samples showed no detectable signal for phospho-JNK. In contrast to JNK, the level of phospho-p38 increased over the 4-h recovery period, most notably in the 60- and 90-min heat-shocked cultures.
|
Further experiments revealed a difference in the duration of JNK phosphorylation in EW36 cultures exposed to heat shock alone or in the presence of SKF (Fig. 6A). A comparison of phospho-JNK1/2 levels during the first 4 h following heat shock revealed that SKF-treated cells had a prolonged period of JNK phosphorylation compared with cells treated with hyperthermia alone (Fig. 6A
; hs SKF compared with hs + SKF). Importantly, one of the kinases immediately upstream of JNK, SEK1, showed an identical pattern of transient phosphorylation following heat shock that was prolonged in the presence of the p38 inhibitor. Moreover, we found that the phosphorylation of SEK1 closely paralleled that of JNK in sensitized EW36 cells (Fig. 6B
). Sensitized cultures showed a progressive lowering of the threshold concentration of arsenite required for phosphorylation of both kinases at 2 h, along with induction of c-Jun at 24 h, after treatment with arsenite alone (As), pretreatment with SKF (SKF + As), or pretreatment with heat shock and SKF (hs + SKF + As). Together, these data indicate that persistent activation of the JNK pathway is involved in sensitization of the resistant BL cell line, in a manner associated with upstream signaling events.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we found that the sensitive BL cell line ST486 showed differences in the activation of specific components of the MAP kinase signal transduction pathways compared with the relatively resistant EW36 cell line. Specifically, our data show that only ST486 cells undergo arsenite-induced apoptosis in the absence of JNK pathway activation. This is evidenced at the lower concentrations of arsenite at which morphological apoptosis and PARP cleavage were detected in the absence of JNK phosphorylation and c-Jun induction. At higher arsenite concentrations, phospho-JNK was detected, but apoptosis occurred rapidly, prior to the induction of c-Jun. This contrasts with the EW36 cell line, which showed clear concentration- and time-dependent association of JNK phosphorylation and c-Jun induction with PARP cleavage and morphological apoptosis. Importantly, even in sensitized EW36 cells, in which cell death was induced at the lower arsenite concentrations, apoptosis was always associated with JNK/c-Jun activation/induction. In addition, our data suggest a requirement for activation of JNK and the subsequent induction of c-Jun, or other downstream products of this pathway, in arsenite-induced apoptosis in EW36 cells, as the AP-1 inhibitor oleandrin blocked both c-Jun induction and PARP cleavage.
Sodium arsenite has multiple targets in cells, with the ability to directly affect mitochondrial function, increase levels of reactive oxygen species, and induce protein damage, primarily by interaction with sulfhydryl groups (Cavigelli et al., 1996; Chen et al., 1998
; Larochette et al., 1999
). At present, we do not know which of these potentially cytotoxic activities is primarily responsible for activation of JNK in the BL cell lines, nor do we know the pathways involved in the JNK-independent induction of apoptosis in ST486 cells. However, it is possible that ST486 cells are susceptible to mitochondrial effects of arsenite, whereas EW36 cells, which overexpress Bcl-2, are not. We have previously shown that ST486 cells rapidly undergo mitochondrial depolarization followed by apoptosis induction after exposure to several mitochondrial toxicants, including compounds that inhibit electron transport or uncouple oxidative phosphorylation (O'Brien et al., 2001
). In contrast, the EW36 cell line was refractory to mitochondrial depolarization and resistant to apoptosis by these agents. Similar to low concentrations of arsenite, apoptosis induction by mitochondrial toxicants in ST486 cells occurred in the absence of JNK phosphorylation. Hence, it is possible that EW36 cells are refractory to the mitochondrial effects of arsenite, but sensitive to its other activities, such as the induction of protein damage or the generation of reactive oxygen speciesconditions that can potentially activate the JNK pathway.
Although some apoptosis-inducing treatments have been reported to downregulate Bcl-2 or inactivate it by phosphorylation (Akao et al., 1998; Hossain et al., 2000
; Srivastava et al., 1999
), we did not observe a change in Bcl-2 levels or in the electrophoretic mobility of the Bcl-2 protein following treatment of EW36 cells with arsenite. In addition, we did not detect changes in the ratio of Bax/Bcl-2 following treatment by arsenite alone or in combination with any of the sensitizing treatments (data not shown). Thus, if JNK-pathway activation is responsible for overcoming Bcl-2mediated resistance in EW36 cells, it does not appear to do so by directly inactivating this protein or affecting the Bax/Bcl-2 ratio.
We found that treatment with nonlethal hyperthermia sensitized EW36 cells to apoptosis induction by subsequent exposure to arsenite, effectively lowering the threshold concentration required for morphological apoptosis and for PARP cleavage. Published studies show that many cell lines, when subjected to a prior nonlethal heat shock, acquire tolerance to subsequent, normally lethal heat stress in addition to treatment with a variety of chemicals. However, such acquisition of thermotolerance depends on the accumulation of stress proteins such as Hsp72 and Hsp27 (Gabai et al., 1997, 2000
; Mosser et al., 1997
; Samali and Cotter, 1996
; Samali et al., 2001
). In our study, EW36 cells were exposed to arsenite shortly after heat shock, prior to the accumulation of high levels of stress proteins, but during a period of transient JNK phosphorylation. This transient activation of JNK after heat shock alone was not sufficient to induce elevated levels of c-Jun protein or to induce apoptosis. However, our data suggest that the introduction of a second stress (such as sodium arsenite, which also activates JNK) pushed the cells over a threshold, resulting in sustained activation of the pathway. This enforced activation of JNK is likely responsible for the synergistic effect of the two stresses on apoptosis induction. Moreover, it is consistent with studies that show that the duration of JNK activation, as opposed to its magnitude of induction, is a key factor in determining whether cells undergo apoptosis by numerous stimuli, including exposure to UV irradiation, tumor necrosis factor, and hyperthermia (Chen et al., 1996
; Volloch et al., 2000
; Westwick et al., 1994
).
Induction of phospho-JNK in cells may be achieved in two ways: by activation of upstream kinases or by inhibition of JNK phosphatases. Arsenite has been reported to act in both ways, with several upstream kinases serving as potential targets (Cavigelli et al., 1996; Hossain et al., 2000
; Meriin et al., 1999
; Porter et al., 1999
), and with a sulfhydryl-containing JNK phosphatase being especially sensitive to inhibition by this toxicant (Cavigelli et al., 1996
). In contrast to arsenite, published studies show that the primary mechanism underlying JNK pathway activation by hyperthermia is mediated by inhibition of one or more JNK phosphatases. Such phosphatase inhibition results in a reduction in the rate of JNK dephosphorylation and subsequent accumulation in the levels of phospho-JNK (Meriin et al., 1999
; Palacios et al., 2001
). Consequently, these increased phospho-JNK levels are observed after heat shock in the absence of increased phosphorylation of upstream kinases.
At present, we cannot exclude the possibility that the activity of a JNK phosphatase is in part responsible for the transient nature of JNK phosphorylation following heat shock in EW36 cells. Indeed, we did observe an inverse relationship between the reduction in the level of phospho-JNK and increase in the level of Hsp72 during the recovery period. Among the many antiapoptotic functions described for Hsp72 is its ability to stabilize JNK phosphatase (Gabai et al., 2000; Yaglom et al., 1999
). Thus, an increase in Hsp72 may lead to the stabilization of JNK phosphatase, along with a concurrent reduction in the level of phosphorylated kinase. However, our data also provide evidence for heat shock-induced activation of the JNK pathway via upstream signals as well.
We found that at least one of the kinases immediately upstream of JNK, SEK1, is phosphorylated in EW36 cells following exposure to hyperthermia as well as to arsenite. Importantly, as for JNK, SEK1 was only transiently activated after heat shock in EW36; pretreatment of EW36 cells with the p38 inhibitor SKF prolonged the period of JNK and SEK1 phosphorylation similarly. Thus, our data suggest that B-lineage cells may differ from other cell types, in that upstream signals are involved in activation of the JNK pathway by hyperthermia. Furthermore, sensitization of cells by either heat shock, p38 kinase inhibition, or the combined treatment, also involves upstream signaling events that lead to the persistent activation of the JNK pathway, along with the progressive lowering of the threshold of arsenite required for SEK1 and JNK phosphorylation and c-Jun induction.
The nature of the upstream signal responsible for the activation of SEK/JNK is presently unknown. Possibilities include the generation of reactive oxygen species and/or the activation of cellular death receptors. Indeed, reactive oxygen species are known inducers of the JNK pathway, and hyperthermia has been shown to induce apoptosis in hematopoietic cells by increasing the production of ligands for Fas and tumor necrosis factor (Farris et al., 1998; Katschinski et al., 1999
). The potential contribution of these pathways in JNK activation and apoptosis induction in BL cells is a subject for further investigation, as is the mechanism underlying the enhancement of JNK pathway activation by inhibition of p38.
Like the JNK pathway, the p38 pathway is commonly associated with induction of apoptosis. However, several studies show that its inhibition may also result in an increase in apoptosis. Inhibition of p38 in Jurkat cells with SB2 also potentiated apoptosis induced by Fas ligand or UV irradiation (Nemoto et al., 1998). By overexpressing different p38 isoforms, that study showed that the antiapoptotic activity of this pathway was attributed to p38b. However, the potential involvement of the JNK pathway was not addressed. In addition, the different isoforms of p38 have been shown to either inhibit or potentiate apoptosis in cardiomyocytes and in other cell types (Wang et al., 1998
; Zechner et al., 1998
). Our data suggest that the p38 pathway itself may be part of a stress response that, when inhibited, sensitizes cells to apoptosis induction. The low but detectable level of p38 activity we observed even in unstressed cells may be a target for sensitization mediated by SKF treatment alone, which is further enhanced by additional stresses that activate this pathway.
Understanding the basis for cellular variation in susceptibility or resistance to apoptosis induction and identifying novel pathways in which this sensitivity may be modulated is important to assess the consequences of chemical exposure and to identify ways in which cells are sensitized to chemically induced apoptosis. Several studies have identified the JNK pathway as an important component of arsenite-induced signaling and apoptosis induction. However, many of these studies used concentrations substantially higher than those used here. Our study shows that discrete thresholds exist for the activation of proapoptotic signaling pathways in B-lineage cells that can be revealed only at lower arsenite concentrations. Moreover, our data invoke the contribution of JNK-dependent and JNK-independent pathways to apoptosis induction in BL cells and the underscore the utility of the BL cell lines for investigating the roles of these pathways in regulating the sensitivity of B-lineage lymphocytes to chemically induced apoptosis and other cytotoxic effects.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akao, Y., Mizoguchi, H., Kojima, S., Naoe, T., Ohishi, N., and Yagi, K. (1998). Arsenic induces apoptosis in B-cell leukaemic cell lines in vitro: Activation of caspases and down-regulation of Bcl-2 protein. Br. J. Haematol. 102, 10551060.[ISI][Medline]
Barchowsky, A., Roussel, R. R., Klei, L. R., James, P. E., Ganju, N., Smith, K. R., and Dudek, E. J. (1999). Low levels of arsenic trioxide stimulate proliferative signals in primary vascular cells without activating stress effector pathways. Toxicol. Appl. Pharmacol. 159, 6575.[ISI][Medline]
Bernstam, L., and Nriagu, J. (2000). Molecular aspects of arsenic stress. J. Toxicol. Environ. Health B Crit. Rev. 3, 293322.[ISI][Medline]
Buzzard, K. A., Giaccia, A. J., Killender, M., and Anderson, R. L. (1998). Heat shock protein 72 modulates pathways of stress-induced apoptosis. J. Biol. Chem. 273, 1714717153.
Burns, L. A., Sikorski, E. E., Saady, J. J., and Munson, A. E. (1991). Evidence for arsenic as the immunosuppressive component of gallium arsenide. Toxicol. Appl. Pharmacol. 110, 157169.[ISI][Medline]
Cavigelli, M., Li, W. W., Lin, A., Su, B., Yoshioka, K., and Karin, M. (1996). The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase. EMBO J. 15, 62696279.[Abstract]
Chen, Y. C., Lin, S. S. Y., and Lin, J. K. (1998). Involvement of reactive oxygen species and capsase 3 activation in arsenite-induced apoptosis. J. Cell. Physiol. 177, 324333.[ISI][Medline]
Chen, Y.-R., Wang, X., Templeton, D., Davis, R. J., Tan, T.-H. (1996). The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and gamma radiation. J. Biol. Chem. 271, 3192931936.
Descotes, J. (1988). Immunotoxicity of Drugs and Chemicals, 2nd ed, p. 444. Elsevier: Amsterdam.
Engel, R. R., Hopenhayn-Rich, C., Receveur, O., and Smith, A. H. (1994). Vascular effects of chronic arsenic exposure: A review. Epidemiol. Rev. 16, 184209.[ISI][Medline]
Fan, S., el-Deiry, W. S., Bae, I., Freeman, J., Jondle, D., Bhatia, K., Fornace, A. J., Jr., Magrath, I., Kohn, K. W., and O'Connor, P. M. (1994). p53 gene mutations are associated with decreased sensitivity of human lymphoma cells to DNA damaging agents. Cancer Res. 54, 58245830.[Abstract]
Farris, M., Latinis, K. M., Kempiak, S. J., Koretzky, G. A., and Nel, A. (1998). Stress-induced Fas ligand expression in T cells is mediated through a MEK kinase 1-regulated response element in the Fas ligand promoter. Mol. Cell. Biol. 18, 54145424.
Gabai, V., Meriin, A. B., Mosser, D. D., Caron, A. W., Rits, S., Shifrin, V. I., and Sherman, M. Y. (1997). Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance. J. Biol. Chem. 272, 1803318037.
Gabai, V., Yaglom, J. A., Volloch, V., Meriin, A. B., Force, T., Koutroumanis, M., Massie, B., Mosser, D., and Sherman, M. Y. (2000). Hsp72-mediated suppression of c-Jun N-terminal kinase is implicated in development of tolerance to caspase-independent cell death. Mol. Cell Biol. 20, 68266836.
Gaidano, G., Ballerini, P., Gong, J. Z., Inghirami, G., Neri, A., Newcomb, E. W., Magrath, I. T., Knowles, D. M., and Dalla-Favera, R. (1991). p53 mutations in human lymphoid malignancies: Association with Burkitt lymphoma and chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. U.S.A. 88, 54135417.[Abstract]
Hall-Jackson, C. A., Goedert, M., Hedge, P., and Cohen, P. (1999). Effect of SB 203580 on the activity of c-Raf in vitro and in vivo. Oncogene 18, 20472054.[ISI][Medline]
Hornung, M., Lindemann, D., Kraus, C., Peters, A., and Berberich, I. (1998). The CD40 TRAF member interacting motif carries the information to rescue WEHI 231 from anti-IgM-induced growth arrest. Eur. J. Immunol28, 38123823.[ISI][Medline]
Hossain, K., Akhand, A. A., Kato, M., Du, J., Takeda, K., Wu, J., Takeuchi, K., Liu, W., Suzuki, H., and Nakashima, I. (2000). Arsenite induces apoptosis of murine T-lymphocytes through membrane raft-linked signaling for activation of c-Jun amino-terminal kinase. J. Immunol. 165, 42904297.
Huang, C., Ma, W.-Y., Li, J., and Dong, Z. (1999a). Arsenic induces apoptosis through a c-Jun NH2-terminal kinase-dependent, p53-independent pathway. Cancer Res. 59, 30533058.
Huang, C., Ma, W.-Y., Li, J., Goranson, A., and Dong, Z. (1999b). Requirement of ERK, but not JNK, for arsenite-induced cell transformation. J. Biol. Chem. 274, 1459514601.
Kaptein, J. S., Lin, C.-K., Wang, C. L., Nguyen, T. T., Kalunta, C. I., Park, E., Chen, F.-S., and Lad, P. M. (1996). Anti-Ig-M-mediated regulation of c-myc and its possible relationship to apoptosis. J. Biol. Chem. 271, 1887518884.
Katschinski, D. M., Robins, H. I., Schad, M., Frede, S., and Fandrey, J. (1999). Role of tumor necrosis factor alpha in hyperthermia-induced apoptosis of human leukemia cells. Cancer Res. 59, 34033410.
Larochette, N., Decaudin, D., Jacotot, E., Brenner, C., Marzo, I., Susin, S. A., Zamzami, N., Xie, Z., Reed, J., and Kroemer, G. (1999). Arsenite induces apoptosis via a direct effect on the mitochondrial permeability transition pore. Exp. Cell Res. 249, 413421.[ISI][Medline]
Lee, Y.-J., and Shacter, E. (1997). Bcl-2 does not protect Burkitt's lymphoma cells from oxidant-induced cell death. Blood 89, 44804492.
Lin, C.-K., Nguyen., T. T., Morgan, T. L., Mei, R.-L., Kaptein, J. S., Kalunta, C. I., Yen, C. F., Park, E., Zou, H. Y., and Lad, P. M. (1998). Apoptosis may be either suppressed or enhanced with strategic combinations of antineoplastic drugs or anti-IgM. Exp. Cell Res. 244, 113.[ISI][Medline]
Liu, J., Kadiiska, M. B., Liu, Y., Lu, T., Qu, W., and Waalkes, M. P. (2001). Stress-related gene expression in mice treated with inorganic arsenicals. Toxicol. Sci. 61, 314320.
Ludwig, S., Hoffmeyer, A., Goebeler, M., Kilian, K., Hafner, H., Neufeld, B., Han, J., and Rapp, U. R. (1998). The stress inducer arsenite activates mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 via a MAPK kinase 6/p38-dependent pathway. J. Biol. Chem. 273, 19171922.
Manna, S. K., Sah, N. K., Newman, R. A., Cisneros, A., and Aggarwal, B. B. (2000). Oleandrin suppresses activation of nuclear transcription factor-B, activator protein-1 and c-Jun N-terminal kinase. Cancer Res. 60, 38383847.
Meriin, A. B., Yaglom, J. A., Gabai, V. L., Mosser, D. D., Goniatsos, S., Zon, L., and Sherman, M. Y. (1999). Protein-damaging stresses activate c-Jun N-terminal kinase via inhibition of its dephosphorylation: A novel pathway controlled by HSP72. Mol. Cell Biol. 19, 25472555.
Merino, R., Ding, L., Veis, D. J., Korsmeyer, S. J., and Nunez, G. (1994). Developmental regulation of the Bcl-2 protein and susceptibility to cell death in B lymphocytes. EMBO J. 13, 683691.[Abstract]
Mosser, D. D., Caron, A. W., Bourget, L., Denis-Larose, C., and Massie, B. (1997). Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis. Mol. Cell. Biol. 17, 53175327.[Abstract]
Muscarella, D. E., and Bloom, S. E. (1997). Involvement of gene-specific DNA damage and apoptosis in the differential toxicity of mitomycin C analogs towards B-lineage versus T-lineage lymphoma cells. Biochem. Pharmacol. 53, 811822.[ISI][Medline]
Muscarella, D. E., Rachlinski, M. K., Sotiriadis, J., and Bloom, S. E. (1998). Contribution of gene-specific lesions, DNA-replication-associated damage, and subsequent transcriptional inhibition in topoisomerase inhibitor-mediated apoptosis in lymphoma cells. Exp. Cell Res. 238, 155167.[ISI][Medline]
Nemoto, S., Xiang, J., Huang, S., and Lin, A. (1998). Induction of apoptosis by SB202190 through inhibition of p38b mitogen-activated protein kinase. J. Biol. Chem. 273, 1641516420.
O'Brien, K. A., Muscarella, D. E., and Bloom, S. E. (2001). Differential induction of apoptosis and MAP kinase signaling by mitochondrial toxicants in drug-sensitive compared to drug-resistant B-lineage lymphoid cell lines. Toxicol. Appl. Pharmacol. 174, 245256.[ISI][Medline]
Palacios, C., Collins, M. K. L., and Perkins, G. R. (2001). The JNK phosphatase M3/6 is inhibited by protein-damaging stress. Curr. Biol. 11, 14391443.[ISI][Medline]
Porter, A. C., Fanger, G. R., and Vaillancourt, R. R. (1999). Signal transduction pathways regulated by arsenate and arsenite. Oncogene 18, 77947802.[ISI][Medline]
Sakata, N., Kawasome, H., Terada, N., Gerwins, P., Johnson, G. L., and Gelfand, E. W. (1999). Differential activation and regulation of mitogen-activated protein kinases through the antigen receptor and CD40 in human B cells. Eur. J. Immunol. 29, 29993008.[ISI][Medline]
Sakata, N., Patel, H. R., Terada, N., Aruffo, A., Johnson, G. L., and Gelfand, E. W. (1995). Selective activation of c-Jun kinase mitogen-activated protein kinase by CD40 on human B cells. J. Biol. Chem. 270, 3082330828.
Samali, A., and Cotter, T. G. (1996). Heat shock proteins increase resistance to apoptosis. Exp. Cell Res. 223, 163170.[ISI][Medline]
Samali, A., Robertson, J. D., Peterson, E., Manero, F., van Zeijl, L., Paul, C., Cotgreave, I. A., Arrigo, A.-P., and Orrenius, S. (2001). Hsp27 protects mitochondria of thermotolerant cells against apoptotic stimuli. Cell Stress Chaperones 6, 4958.[ISI][Medline]
Smith, P. C., and Sladek, N. E. (1985). Sensitivity of murine B- and T-lymphocytes to oxazaphosphorine and non-oxazaphosphorine nitrogen mustards. Biochem. Pharmacol. 34, 34593463.[ISI][Medline]
Sommers, S. C., and McManus, R. G. (1953). Multiple arsenical cancers of skin and internal organs. Cancer (Phila.) 6, 347359.[ISI]
Srivastava, R. K., Mi, Q.-S., Hardwick, M. H., and Longo, D. L. (1999). Deletion of the loop region of Bcl-2 completely blocks paclitaxel-induced apoptosis. Proc. Natl. Acad. Sci. U.S.A. 96, 37753780.
Trouba, K. J., Wauson, E. M., and Vorce, R. L. (2000). Sodium arsenite-induced dysregulation of proteins involved in proliferative signaling. Toxicol. Appl. Pharmacol. 164, 161170.[ISI][Medline]
Uckun, F. M., Mitchell, J. B., Oroz, U., Park, C. H., Waddick, K., Fredman, N., Oubaha, L., Min, W. S., and Song, C. W. (1991). Radiation sensitivity of human B-lineage lymphoid precursor cells. Int. J. Radiat. Oncol. Biol. Phys. 21, 15531560.[ISI][Medline]
Volloch, V., Gabai, V. L., Rits, S., Force, T., and Sherman, M. Y. (2000). Hsp72 can protect cells from heat-induced apoptosis by accelerating the inactivation of stress kinase JNK. Cell Stress Chaperones 5, 139147.[ISI][Medline]
Waalkes, M. P., Fox, D. A., States, J. C., Patierno, S. R., and McCabe, M. J., Jr. (2000). Metals and disorders of cell accumulation: Modulation of apoptosis and cell proliferation. Toxicol. Sci. 56, 255261.
Wang, Y., Huang, S., Sah, V. P., Ross, J., Jr., Brown, J. H., Han, J., and Chien, K. R. (1998). Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family. J. Biol. Chem. 273, 21612168.
Westwick, J. K., Weitzel C., Minden, A., Karin, M., and Brenner, D. A. (1994). Tumor necrosis factor alpha stimulates AP-1 activity through prolonged activation of the c-Jun kinase. J. Biol. Chem. 269, 2639626401.
Wilmer, J. L., Colvin, O. M, and Bloom, S. E. (1992). Cytogenetic mechanisms in the selective toxicity of cyclophosphamide analogs and metabolites towards avian embryonic B lymphocytes in vivo. Mutat. Res. 268, 115130.[ISI][Medline]
Yaglom, J. A., Gabai, V. L., Meriin, A. B., Mosser, D. D., and Sherman, M. Y. (1999). The function of HSP72 in suppression of c-Jun N-terminal kinase activation can be dissociated from its role in prevention of protein damage. J. Biol. Chem. 274, 2022320228.
Young, P. R., McLaughlin, M. M., Kumar, S., Kassis, S., Doyle, M. L., McNulty, D, Gallagher, T. F., Fisher, S., McDonnell, P. C., Carr, S. A., Huddleston, M. J., Seibel, G., Porter, T. G., Livi, G. P., Adams, J. L., and Lee, J. C. (1997). Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site. J. Biol. Chem. 272, 1211612121.
Zechner, D., Craig, R., Hanford, D. S., McDonough, P. M., Sabbadini, R. A., and Glembotski, C. C. (1998). MKK6 activates myocardial cell NF-kappaB and inhibits apoptosis in a p38 mitogen-activated protein kinase-dependent manner. J. Biol. Chem. 273, 82328239.