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Cell Growth & Differentiation Vol. 12, 137-145, March 2001
© 2001 American Association for Cancer Research

Activation of c-Jun NH2-Terminal Kinase/Stress-activated Protein Kinase (JNK/SAPK) Is Critical for Hypoxia-induced Apoptosis of Human Malignant Melanoma1

Manfred Kunz2, Saleh Ibrahim, Dirk Koczan, Hans-J. Thiesen, Hans J. Köhler, Till Acker, Karl H. Plate, Stephan Ludwig, Ulf R. Rapp, Eva-B. Bröcker, Goos N. P. van Muijen, Egbert Flory and Gerd Gross

Department of Dermatology and Venereology [M. K., G. G.] and Institute of Immunology [S. I., D. K., H-J. T., H. J. K.], University of Rostock, 18055 Rostock, Germany; Department of Neuropathology, University of Erlangen-Nürnberg, 91054 Erlangen, Germany [T. A., K. H. P.]; Institut für Medizinische Strahlenkunde und Zellforschung [S. L., U. R. R.] and Department of Dermatology [E-B. B.], University of Würzburg, 97080 Würzburg, Germany; Department of Pathology, University of Nijmegen, 6500 Nijmegen, the Netherlands [G. N. P. v. M.]; and Paul-Ehrlich-Institute, 63225 Langen, Germany [E. F.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Mitogen-activated protein kinase (MAPK) signaling was examined in malignant melanoma cells exposed to hypoxia. Here we demonstrate that hypoxia induced a strong activation of the c-Jun NH2-terminal kinase (JNK), also termed stress-activated protein kinase (SAPK), in the melanoma cell line 530 in vitro. Other members of the MAPK family, e.g., extracellular signal-regulated kinase and p38, remained unaffected by the hypoxic stimulus. Activated JNK/SAPK could also be observed in the vicinity of hypoxic tumor areas in melanoma metastases as detected by immunohistochemistry. Functional analysis of JNK/SAPK activation in the melanoma cell line 530 revealed that activation of JNK/SAPK is involved in hypoxia-mediated tumor cell apoptosis. Both a dominant negative mutant of JNK/SAPK (SAPKß K->R) and a dominant negative mutant of the immediate upstream activator of JNK/SAPK, SEK1 (SEK1 K->R), inhibited hypoxia-induced apoptosis in transient transfection studies. In contrast, overexpression of the wild-type kinases had a slight proapoptotic effect. Inhibition of extracellular signal-regulated kinase and p38 pathways by the chemical inhibitors PD98058 and SB203580, respectively, had no effect on hypoxia-induced apoptosis. Under normoxic conditions, no influence on apoptosis regulation was observed after inhibition of all three MAPK pathways. In contrast to recent findings, JNK/SAPK activation did not correlate with Fas or Fas ligand (FasL) expression, suggesting that the Fas/FasL system is not involved in hypoxia-induced apoptosis in melanoma cells. Taken together, our data demonstrate that hypoxia-induced JNK/SAPK activation appears to play a critical role in apoptosis regulation of melanoma cells in vitro and in vivo, independent of the Fas/FasL system.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The local growth of malignant tumors is largely dependent on adequate nutrient and oxygen supply (1) . Rapidly growing metastases of these tumors are exposed to hypoxic or even anoxic conditions, at least in the central part of the tumor. Tissue hypoxia acts as a strong inducer of angiogenic factors in a variety of malignant tumors, e.g., glioblastoma (2 , 3) . Angiogenic factors such as vascular endothelial growth factor, fibroblast growth factor, and interleukin 8 induce neovascularization and help to overcome the adverse conditions of the microenvironment (4) . Interestingly, even after neovascularization, tumor areas remain under low oxygen tension due to inadequate vasculature after neoangiogenesis (5) . Hypoxia has a profound influence on tumors because it leads to higher proliferation rates and enhanced metastatic potential (6 , 7) , designated as higher aggressiveness. Therefore, tumor cell responses to hypoxia are of central importance for the understanding of tumor progression. Initial attempts to characterize the transcriptional regulation of hypoxia-inducible genes (e.g., angiogenic factors) were made (8 , 9) . However, few data exist about possible upstream activators of transcription factors under hypoxic stress (10, 11, 12) .

Extracellular stimuli that exert influence on gene expression are mediated by several parallel organized signal-transducing cascades (for review, see Ref. 13 ). One of the most intensively studied signaling pathways is the mitogenic Raf/MEK3 /ERK kinase cascade that responds to growth and differentiation-inducing factors such as epidermal growth factor and platelet-derived growth factor (14) . Recently, parallel kinase cascades have been identified that respond to extracellular stresses such as inflammatory cytokines interleukin 1, TNF-{alpha}, cellular injury (heat, UV, and ionizing irradiation), and osmotic shock (for review, see Refs. 15, 16, 17, 18 ). Members of these pathways are the SAPKs (also termed JNKs) and p38. Because hypoxia is a typical stress factor for rapidly growing tumor cells, it is tempting to speculate that hypoxia induces JNK/SAPK and p38 activation in this cell type. JNK/SAPK activation had been shown in rat cardiac myocytes after hypoxia/reoxygenation (11 , 12) . However, data about the physiological relevance of JNK/SAPK activation are still lacking (19) . Furthermore, the participation of upstream activators of the mentioned kinase remains to be elucidated.

In the present report, we examined the different signaling pathways under hypoxic conditions in human malignant melanoma cells and analyzed the functional significance of activated kinases for apoptosis regulation. In vitro kinase assays were performed to analyze the three major MAPK signaling pathways, namely, the ERK, JNK/SAPK, and p38 pathways. It is demonstrated that hypoxia induces activation of the JNK/SAPK pathway but not the ERK and p38 pathways. Dominant negative mutants of JNK/SAPK and SEK1 (SEK1 acts as an immediate upstream activator of JNK/SAPK) inhibited hypoxia-induced apoptosis, whereas wild-type JNK/SAPK had a slight proapoptotic effect. Under normal oxygen tension, wild-type and dominant negative kinases had no significant effect on apoptosis. The present data give new insights into the molecular stress response of tumor cells and apoptosis control under tissue hypoxia that might have implications for the development of future therapeutical strategies.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Hypoxic Induction of JNK/SAPK in 530 Melanoma Cells in Vitro.
Melanoma cells were exposed to hypoxia for 1, 3, and 24 h, respectively. Parallel cultures were constantly kept under normal oxygen tension. JNK/SAPK, p38, and ERK activity was determined by immunocomplex kinase assays using GST c-Jun for JNK/SAPK, recombinant 3pK protein for p38, and MBP for ERK, respectively, as substrates (20 , 21) . Fig. 1Citation shows a representative of four independent experiments. Hypoxia induced a strong up-regulation (5-fold) of JNK/SAPK activity after 3 h. Further exposure to hypoxia led to a down-regulation of JNK/SAPK activity, indicating that JNK/SAPK activation was transient in these experiments. A slight (2-fold) up-regulation was observed for ERK activity after 3 h of hypoxia. However, this is regarded as not significant. p38 activity was generally low and was not inducible by hypoxia. Immunoblots of kinases are shown to confirm equal loading of immunoprecipitated protein in SDS-PAGE. Together, these data demonstrate that hypoxia specifically activates the JNK/SAPK signaling pathway, whereas both other pathways examined remained unaffected.



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Fig. 1. Immunocomplex kinase assays and Western blots of JNK/SAPK, p38, and ERK under normoxia and hypoxia in melanoma cell line 530. Immunoprecipitated kinases were analyzed at different time points after exposure of melanoma cell line 530 to hypoxia. In vitro kinase assays were performed in kinase buffer supplemented with 5 µCi of [{gamma}-32P]ATP, 0.1 mM ATP, and substrate proteins at 30°C for 15 min. JNK/SAPK, ERK, and p38 activity was assayed with GST-cJun, MBP, and 3pK (K/M) as substrates, respectively. Proteins were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes. The intensity of radioactively labeled bands was determined by phosphorimaging. Every experiment was repeated four times. Hypoxia induced up-regulation (5-fold) of JNK/SAPK activity after 3 h. Further exposure to hypoxia led to a down-regulation of JNK/SAPK.

 
Expression of Activated JNK/SAPK in Melanoma Metastases with Necrotic/Hypoxic Areas.
To further address the question whether our in vitro finding might be reflected by the in vivo situation, activated kinases were analyzed in necrotic/hypoxic melanoma metastases. For this purpose, tissue sections of necrotic melanoma metastases were stained for the activated MAPKs JNK/SAPK, ERK (Ab termed anti-active MAPK), and p38. A strong staining for the activated stress kinase, phospho-JNK/SAPK, could be observed in close vicinity to necrotic/hypoxic areas (Fig. 2, A and B)Citation , whereas the necrotic area itself (depicted as N) and histopathologically normal-appearing areas (data not shown) displayed negative staining. The presence of hypoxia in the phospho-JNK/SAPK-positive tumor areas was confirmed by staining of serial sections for HIF-1{alpha} (Fig. 2, C and D)Citation . The staining pattern of both phospho-JNK/SAPK and HIF-1{alpha} largely overlapped in all tested melanoma metastases. CD34-positive vessels were detected at a distance far from the hypoxic areas in the connective tissue surrounding the tumor (Fig. 2, E and F)Citation . ERK and p38 activation could not be detected in clear association with necrotic/hypoxic areas of melanoma metastases. However, isolated positively stained cells were detected scattered throughout the tumor (data not shown). In contrast to the melanoma metastases, all benign melanocytic nevi were negative for activated JNK/SAPK (data not shown), and, both activated kinases, ERK and p38, were also not detected in benign nevi. Due to its association with areas of necrosis, activated (phosphorylated) JNK/SAPK appears to play a role in necrotic/hypoxic tumor areas.



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Fig. 2. Immunohistochemical staining of necrotic/hypoxic melanoma metastases for the activated kinase JNK/SAPK. A and B, immunohistochemical staining of phospho-JNK/SAPK in a melanoma metastasis. Arrowheads indicate the demarcation line of positively stained tumor cells adjacent to a necrotic tumor area depicted as N. B, higher power view of A. C and D, immunohistochemical staining of a serial section of the metastasis from A and B for HIF-1{alpha} shows positive staining in the same tumor area. D, higher power view of C. E and F, immunohistochemical detection of blood vessels (anti-CD34 staining) in the same metastasis at a distance far from the necrotic area. F, higher power view of E. Original magnification in A, C, and E, x200; original magnification in B, D, and F, x400. Scale bars in A, C, and E = 50 µm; scale bars in B, D, and F = 25 µm. N, necrotic area.

 
Role of JNK/SAPK and SEK1 in Hypoxia-induced Apoptosis in Melanoma Cells.
JNK/SAPK activation has been implicated in the regulation of the apoptotic process in various cell types (22, 23, 24, 25, 26) . Here we used a dominant negative approach to analyze the role of hypoxia-induced JNK/SAPK activity for melanoma cell apoptosis. Melanoma cells were transiently transfected with wild-type SAPKß54, wild-type SEK1, and dominant negative mutants of SAPKß54 and SEK1, respectively. Cells were either kept under normoxia or exposed to hypoxia. Apoptotic cells were analyzed by flow cytometry. Data are given as mean ± SD of four independent experiments, and asterisks indicate statistical significance of reduced apoptosis compared to control experiments (Student’s t test; P <= 0.05 is regarded as statistically significant). After exposure to hypoxia, apoptosis increased over time. Fig. 3, A and BCitation , shows a strong increase of apoptosis (more than 3-fold) in mock-transfected melanoma cells after exposure to 24 h of hypoxia compared to cells under normoxia. Transfection of wild-type or dominant negative kinases (SAPK/SAPK K->R, and SEK1/SEK K->R) had only marginal effects on the rate of apoptotic cells under normoxia. In contrast, transfection of the dominant negative mutants of both kinases exerted an apoptosis-protective effect after both 12 and 24 h of hypoxia (Fig. 3B)Citation . At the same time, a slight proapoptotic effect was observed for wild-type kinases SAPK and SEK1, at least after 12 h of hypoxia. Inhibition of the other MAPK pathways by the chemical inhibitors PD98058 for MEK (upstream activator of ERK) and SB203580 for p38 had no influence on the rate of apoptotic cells under normoxia or hypoxia (Fig. 3, A and B)Citation . Taken together, JNK/SAPK signaling appears to be of critical importance for hypoxia-mediated apoptosis in melanoma cells.



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Fig. 3. FACScan analysis of apoptotic melanoma cells (melanoma cell line 530) after exposure to hypoxia and in vitro luciferase assays of c-Jun transcriptional activity. A and B, apoptotic cells were identified by staining with biotin-conjugated annexin V. Melanoma cells under normoxic conditions and hypoxia (12 h/24 h) were incubated in 200 µl of annexin buffer containing 4 µl of annexin V-biotin for 30 min at 4°C. After that, cells were incubated with streptavidin cytochrome c and analyzed by flow cytometry (FACScan). Cells were transiently transfected with the wild-type or dominant negative interfering kinases of SAPKß54 or SEK1 or exposed to the chemical inhibitors PD98058 or SB203580. Representative FACScan dot plots of one experiment are shown in A. Diagrams in B show the mean ± SD of the percentage of apoptotic melanoma cells from four independent experiments. Asterisks (*, **) indicate the statistical significance of reduced apoptosis after transfection with the dominant negative interfering kinases compared to the wild-type kinases (P <= 0.05). C, in vitro luciferase assays of c-Jun transcriptional activity after transfection of melanoma cells with wild-type and dominant negative interfering kinases (SAPKß54, SAPK K->R, SEK, and SEKK->R), respectively. Asterisks (*, **) indicate the statistical significance of reduced c-Jun-dependent transcriptional activity after transfection with the dominant negative interfering kinases.

 
Dominant Negative JNK/SAPK and SEK Inhibit c-Jun-mediated Transcriptional Activity under Hypoxia.
The inhibitory effects of dominant negative interfering kinases SAPK K->R or SEK K->R on hypoxia-induced kinase activity were analyzed via measurement of the transactivating capacity of the JNK/SAPK downstream target c-Jun. Under normoxic conditions, no statistically significant influence of wild-type or dominant negative interfering kinases on c-Jun-dependent transcription could be observed (Fig. 3C)Citation , presumably due to low constitutive activity of the transfected wild-type kinases and low baseline activity of the endogeneous kinases. However, hypoxia-induced activation was markedly reduced after transfection of the dominant negative interfering kinases SAPK K->R or SEK K->R (Fig. 3C)Citation . Again, the wild-type kinases had no significant effects. Transfection of a MEK kinase-containing plasmid served as a positive control for reporter gene activity and led to a 6–8-fold induction (data not shown). Taken together, it is shown that the transfected kinase constructs SAPK K->R and SEK K->R exerted inhibitory effects on the endogeneous kinases.

Fas/FasL Is Not Involved in Hypoxia-induced Apoptosis in 530 Melanoma Cells.
Because stress-induced activation of JNK/SAPK and apoptosis have been linked to induction of FasL (23 , 25) , we further analyzed whether hypoxia-induced JNK/SAPK activation results in FasL or Fas up-regulation in the melanoma cell line 530. For this purpose, mRNA levels of FasL and Fas were investigated by RT-PCR and real-time PCR (TaqMan analysis). A constitutive high expression of Fas under normoxic and hypoxic (24 h) conditions was observed (Fig. 4)Citation . In contrast, no FasL expression could be detected under both normoxic and hypoxic conditions by RT-PCR. PHA-stimulated Jurkat cells were used as a positive control for FasL and displayed strong FasL expression (Fig. 4)Citation . Real-time PCR analysis was used for quantification of these findings. The melanoma cells displayed high levels of Fas mRNA, which was only slightly induced after hypoxia (24 h) [15,100–17,400 mRNA molecules/100 ng total RNA under normoxia and 18,700–21,750 mRNA molecules/100 ng total RNA upon hypoxia (data not shown)]. In contrast, no FasL mRNA was detected under normal conditions and hypoxia. For FasL, the amount of mRNA molecules/100 ng total RNA was below the cutoff of 100 molecules/100 ng total RNA. To further analyze these data on the protein level, flow cytometry was performed for cell surface expression of both apoptosis molecules on melanoma cells (530 cell line). As shown in Fig. 5Citation , strong surface expression of Fas was detected on melanoma cells under normoxia (Fig. 5A)Citation and remained unchanged after 24 h of hypoxia (Fig. 5B)Citation . FasL expression was negative in 530 melanoma cells (Fig. 5C)Citation and was not inducible after exposure to hypoxia (24 h; Fig. 5DCitation ). Thus, protein expression parallels mRNA expression. This argues against a role of both apoptosis molecules, Fas and FasL, in hypoxia-induced apoptosis.



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Fig. 4. RT-PCR of Fas and FasL in 530 melanoma cells under hypoxia. 530 melanoma cells were either kept under normoxia or exposed to 24 h of severe hypoxia. Jurkat cells stimulated with PHA and kept under normal culture conditions served as a positive control for FasL expression. Total RNA was extracted using a commercially available RNA extraction kit. Total RNA (1 µg) was reverse transcribed into cDNA using an oligo(dT)16 primer. For PCR amplification, specific primers for Fas and FasL were used. PCR conditions were as follows: an initial 5-min denaturation step was followed by 35 cycles of 30 s of denaturation at 94°C, 30 s of annealing at 58°C, 1 min of primer extension at 72°C, and a final primer extension step of 10 min at 72°C. PCR products were visualized after electrophoresis on a 1% agarose gel.

 


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Fig. 5. FACScan analysis of Fas and FasL expression in the melanoma cell line 530 under normoxia and hypoxia. 530 melanoma cells were stained with a primary mouse antihuman FasR moAb or mouse antihuman FasL moAb. Incubation with isotype mouse IgG served as a control. After that, cells were washed, and a phycoerythrin-conjugated secondary Ab was added for 30 min at 4°C. Analysis was performed by flow cytometry (FACScan; Becton Dickinson). Ten thousand cells were counted. Isotype control (background staining) is shown as dotted lines.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Rapidly growing tumors and tumor metastases are exposed to hypoxic or even anoxic conditions in the tissue microenvironment. Although this is well known for a variety of tumors, the reaction pattern of tumor cells to hypoxic conditions in the microenvironment and the pathophysiological consequences for tumor growth are still poorly defined. In the present report, it is demonstrated that the stress-activated kinase JNK/SAPK is activated in melanoma cell line 530 under experimentally induced hypoxia. Both the MAPK kinase 6/p38 stress pathway and the Raf/MEK/ERK pathway remained unaffected. It is shown by transient transfection studies with wild-type and dominant negative SAPKß and dominant negative SEK1 that the activated JNK/SAPK pathway is critical for hypoxia-induced apoptosis in these cells.

The SAPK JNK/SAPK had been first described as a c-Jun kinase responding to a variety of stimuli, e.g., heat shock, protein synthesis inhibitors (cycloheximide and anisomycin), and TNF-{alpha}, but was not activated by mitogenic stimuli (16) . Although little is known about the physiological role and downstream targets of the stress signaling pathways (19) , there is increasing evidence that JNK/SAPK signaling plays a role in the process of apoptosis (26) . However, there is still controversy regarding whether activated JNK/SAPK exerts proapoptotic effects or might even be apoptosis protective.

On one hand, there is increasing evidence that activated JNK/SAPK is linked to enhanced apoptosis after a variety of proapoptotic stimuli, e.g., FAS/Apo-1 activation (27) , anticancer drugs (28 , 29) , dopamine (24) , UV irradiation (23) , X-rays, heat, and H202 exposure (22) . On the other hand, data are currently available showing that activated JNK/SAPK might exert a protective function after proapoptotic stimuli. It has been shown that thymocytes of SEK1-deficient mice displayed increased susceptibility to TNF-induced apoptosis (30) . Furthermore, TRAF2-deficient mice showed increased susceptibility to TNF-induced apoptosis, which correlated with a severe reduction in JNK/SAPK activation (31) . Interestingly, despite enhanced apoptosis, nuclear factor {kappa}B activation, which is well known for its role in apoptosis protection (32) , remained unaffected.

As a third alternative, JNK/SAPK activation might occur independently of the apoptosis pathways or might be a consequence rather than a prerequisite for the apoptotic processes (33, 34, 35) . Mosser et al. (34) showed that SAPK activation is not directly linked to apoptosis in a human T-cell leukemia cell line exposed to heat stress. In line with this, recently published data showed that dominant negative mutants of the upstream activator of SAPK, SEK1, blocked JNK/SAPK activation but had no effect on Fas-induced apoptosis (33) . A possible explanation for the obvious heterogeneous findings between apoptosis induction, apoptosis protection, and the lack of direct correlation between activated JNK/SAPK and apoptosis might be due to the different cellular backgrounds and the different stimulatory conditions.

In the present report, it is demonstrated that JNK/SAPK is activated in vitro on exposure of melanoma cells to hypoxia. Further functional analysis revealed that hypoxia-induced apoptosis in vitro was reduced by transient transfection with a dominant negative mutant of JNK/SAPK, whereas overexpression of wild-type JNK/SAPK led to slightly enhanced apoptosis. Essentially the same results were obtained for SEK1. From these data, it is concluded that the JNK/SAPK signaling pathway is critically involved in apoptosis signaling under hypoxia in melanoma cells. Interestingly, inhibition of JNK/SAPK signaling had no effect on apoptosis regulation under normoxia. It might be presumed that a particular intracellular microenvironment influences the role of activated JNK/SAPK. Recent data in fact provide evidence for a cross-talk between JNK/SAPK and the cellular redox potential (36) . It has been shown that the redox-sensitive molecule GST-{pi} is involved in JNK/SAPK activation.

In search of possible downstream targets for activated JNK/SAPK under hypoxia, the influence on the Fas/FasL system was investigated. It could be demonstrated that FasL obviously does not play any role in apoptosis induction under hypoxia in melanoma cell line 530 because FasL mRNA and protein expression was not detectable in these cells by quantitative real time RT-PCR and flow cytometry. These findings are in contrast to recently published data (23 , 25) and further underline the possibility that JNK/SAPK and apoptosis regulation may be cell type and stimulus specific. Other possible downstream targets of JNK/SAPK activation are less well defined and in general comprise the family of c-Jun-dependent genes (37 , 38) .

It is well accepted that tumors become hypoxic beyond a diameter of more than 2–3 mm. Consequently, at late stage growth, especially in metastases, hypoxic areas are a common feature. In accordance with our in vitro studies, in vivo activated JNK/SAPK could be detected in necrotic/hypoxic melanoma metastases. Thus, presumably the same mechanisms of hypoxia-induced JNK/SAPK activation appear to be active in vivo. The pathophysiological relevance of these findings might be that enhanced apoptosis due to hypoxia heralds enhanced, more aggressive tumor growth. It is well known that oncogenically transformed cells show a tight control of proliferation and apoptosis, and there is strong evidence that rapidly proliferating aggressive tumors display enhanced apoptosis (39) . The fact that hypoxia selects for highly aggressive tumor cells via hypoxia-induced apoptosis has been shown recently (7) .

Taken together, we presume that the JNK/SAPK signaling pathway plays an important role in apoptosis regulation under hypoxia in melanoma cells. Because tumor aggressiveness is closely linked to tissue hypoxia, interfering with this pathway might be a target for future therapeutical approaches.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Reagents and Abs
The specific chemical inhibitor PD98058 (Calbiochem, Bad Soden, Germany) for inhibition of MEK activation was used at a concentration of 10 µM; the specific chemical p38 inhibitor SB203580 (Calbiochem) was used at a concentration of 1 µM. Immunohistochemical staining of tissue sections was performed using anti-active JNK rabbit pAb (Promega, Heidelberg, Germany), anti-active MAPK/ERK rabbit pAb (Promega), anti-phospho-p38 MAPK rabbit Ab (New England Biolabs, Beverly, MA), anti-CD34 moAb (Immunotech, Hamburg, Germany), and anti-HIF-1{alpha} moAb (Novus). Immunoprecipitation was performed using Abs raised against ERK2 (sc-154), JNK1 (sc-474), and p38 (sc-535), respectively (all purchased from Santa Cruz Biotechnology, Inc., Heidelberg, Germany).

cDNA Constructs
Wild-type SAPKß54, dominant negative SAPKß54 (termed SAPK K->R), wild-type SEK1, and dominant negative SEK1 (termed SEK K->R), respectively, were used for transient transfection studies (20 , 40) . All were subcloned into eukaryotic expression vector pEBG. Cells were transiently transfected with the vector constructs or empty vector (pEBG) as a control.

Cell Lines, Culture Conditions, and Transfection
The human melanoma cell line 530 (41) was maintained in RPMI 1640 (Linaris, Bettingen, Darmstadt, Germany) supplemented with 10% FCS (Linaris), 2 mM L-glutamine, 100 units/ml penicillin/streptomycin, and 1% nonessential amino acids. Cells were cultured in a humidified incubator (37°C in 5% CO2:95% air) and passaged when confluent. For hypoxic treatment, cultures were transferred to an anaerobic culture chamber (Anaerocult A; Merck, Darmstadt, Germany) for different time peroids as described previously (42 , 43) . Cells were exposed to the indicated times of hypoxia. Parallel cultures were constantly kept under normal oxygen conditions (normoxia). Cell lysates were prepared for immunocomplex kinase assays. For inhibition studies, 530 cells were transfected with 2 µg of the appropriate plasmid DNA of wild-type SAPKß, dominant negative SAPKß (SAPK K->R), wild-type SEK1, and dominant negative SEK1 (SEK K->R) respectively, using the DMRIE-C (1,2-dimyristyloxypropyl-3-dimethyl-hydroxy-ethyl-ammonium-bromide) reagent (Life Technologies, Inc., Eggenstein, Germany) according to the manufacturer’s specifications. Empty pEBG was used for mock transfection. Before transfection, cells were kept under normal culture conditions. In short, 2 µg of plasmid DNA of each plasmid and 10 µl of DMRIE-C, respectively, were diluted in 1 ml of reduced-serum culture medium (OptiMEM; Life Technologies, Inc.). Both mixtures were put together and incubated at room temperature for 30 min with occasional shaking. Medium was removed from cell cultures and replaced by the lipid-DNA complex-containing medium and incubated overnight for additional 24 h. Thereafter, cells were kept in 10% FCS-supplemented RPMI 1640 for 24 h under normal oxygen tension before exposure to hypoxia.

Immunoprecipitation and Western Blot Analysis
530 melanoma cells were lysed in a modified radioimmunoprecipitation buffer [25 mM Tris-HCl (pH 8.0) containing 137 mM NaCl, 10% (v/v) glycerol, 0.1% SDS, 0.5% (v/v) deoxycholate, 1% (v/v) NP40, 2 mM EDTA, 1 mM pefabloc, 1 mM sodium vanadate, 5 mM benzamidine, and 5 µg/ml leupeptin] on ice for 30 min. After centrifugation, supernatants were incubated with anti-ERK, anti-JNK/SAPK, and anti-p38 antisera (Santa Cruz Biotechnology, Inc.) for 2 h at 4°C. The immunocomplexes were precipitated with protein A-agarose and washed with high salt TLB buffer [20 mM Tris (pH 7.4), 50 mM sodium ß-glycerophosphate, 20 mM sodium PPi, 500 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 2 mM EDTA, 1 mM pefabloc, 1 mM sodium orthovanadate, 5 mM benzamidine, 5 µg/ml aprotinin, and 5 µg/ml leupeptin]. Immunoprecipitates were used for immunocomplex kinase assays. For protein detection in Western blots, the immunocomplexes were suspended in electrophoresis sample buffer and heated to 100°C for 3 min. After SDS-PAGE, gels were electroblotted onto polyvinylidene difluoride membranes (Millipore) and subjected to immunodetection using the appropriate primary Ab for ERK, JNK/SAPK, and p38, respectively. Proteins were visualized using horseradish peroxidase-conjugated protein A (Amersham, Braunschweig, Germany) and a standard enhanced chemiluminescence reaction (Amersham).

Immunocomplex Kinase Assays
Immunoprecipitated kinases were washed twice, both in high salt TLB and kinase buffer [10 mM MgCl2, 25 mM ß-glycerophosphate, 25 mM HEPES (pH 7.5), 5 mM benzamidine, 0.5 mM DTT, and 1 mM sodium vanadate], and then assayed in the same buffer supplemented with 5 µCi of [{gamma}-32P]ATP, 0.1 mM ATP, and substrate proteins at 30°C for 15 min. ERK activity was assayed with MBP (Sigma, Deisenhofen, Germany), JNK/SAPK activity was assayed with GST-cJun, and p38 activity was assayed with 3pK [K/M (20 , 21) ] as substrates. Proteins were separated by SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and detected by a Bio Imaging Analyzer (Fuji). Every experiment was repeated at least three times. Intensity of radioactively labeled bands was determined by phosphorimaging (Fujix Bas-2000; Fuji).

Immunohistochemistry
Five-µm paraffin-embedded tissue sections of eight melanoma metastases and eight benign melanocytic nevi (for control purposes), all from different patients, were investigated by immunohistochemistry for activated ERK, JNK/SAPK, p38 kinase, HIF-1{alpha}, and CD34, respectively. All metastases displayed necrotic/hypoxic areas. Tissue sections were fixed in alcohol and rehydrated before staining. Incubation with the primary Ab (anti-active JNK pAb, anti-active MAPK/ERK pAb, anti-phospho-p38 MAPK pAb, and anti-CD34 for blood vessels) was carried out for 30 min at room temperature. Normal mouse IgG was used as a control. Slides were then washed, and in a second incubation step, an IgG antimouse horseradish peroxidase-coupled Ab was used. Staining for HIF-1{alpha} was carried out as described recently (44) . The specific Ab binding was visualized using 0.2 mg/ml 3-amino-9-ethylcarbazole (Sigma) as substrate for horseradish peroxidase. Sections were counterstained slightly with hematoxylin.

Flow Cytometric Analysis
Apoptosis.
Early apoptotic events in melanoma cells in vitro (melanoma cell line 530) were detected by staining with biotin-conjugated annexin V (PharMingen, Hamburg, Germany). Annexin binds to exposed phosphatidylserine on the surface of apoptotic cells (45) . 530 melanoma cells were incubated in 200 µl of annexin buffer containing sterofundin solution (Braun, Melsungen, Germany) supplemented with 2% HEPES (pH 7.3) and 4 µl of annexin-biotin for 30 min at 4°C. After that, cells were incubated with streptavidin cytochrome c (Bender, Heidelberg, Germany). Cells were analyzed by flow cytometry (FACScan; Becton Dickinson, Heidelberg, Germany) using the CellQuest software (Becton Dickinson).

Fas/FasL Expression.
For detection of cell surface expression of the apoptosis molecules Fas and FasL in the melanoma cell line 530, cells were incubated with a primary mouse antihuman FasR moAb (PharMingen) or a mouse antihuman FasL moAb (PharMingen) for 30 min at 4°C. Incubation with isotype mouse IgG was carried out as a control. After that, cells were washed, and a phycoerythrin-conjugated secondary Ab (Dako, Hamburg, Germany) was added for 30 min at 4°C. After washing, analysis was performed by flow cytometry (FACScan; Becton Dickinson). Ten thousand cells were counted, and the percentage of FasR- and FasL-positive cells was determined using the Cell Quest software.

In Vitro Luciferase Assay
To control the effect of wild-type and dominant negative interfering kinases on hypoxia-induced JNK/SAPK activity, reporter gene analyses were performed. A commercially available system (PathDetect trans-reporting system; Stratagene, Heidelberg, Germany) was used that detects JNK/SAPK activity via the transcriptional activity of its downstream target c-Jun. For this purpose, 530 melanoma cell were transfected with 50 ng of the fusion transactivator plasmid, pFA2-cJun, containing the activation domain of c-Jun fused with the yeast GAL4 binding domain and 1 µg of pFR-Luc reporter plasmid carrying five tandem repeats of GAL4 binding sites linked to firefly luciferase gene. Cotransfection was done with wild-type and dominant negative interfering kinases, JNK/SAPK or SEK, and empty vector as a control. Total cell extracts were prepared, and luciferase assays were carried out as described recently (43) using a Berthold luminometer for measurement of luciferase activity (Berthold, Bald Wildbach, Germany). Total protein concentration was measured by the Bradford technique (Bio-Rad, München, Germany). The luciferase activities were normalized on the basis of protein content as well as on ß-gal activity of cotransfected Rous sarcoma virus-ß-gal vector. The ß-gal assay was performed with 20 µl of precleared cell lysate according to a standard protocol, as described previously (40) . The mean ± SD of four independent experiments are shown.

RT-PCR
To determine Fas and FasL mRNA expression in the melanoma cell line 530 under normoxia and hypoxia, total RNA was extracted using a commercially available RNA extraction kit (Rneasy; Qiagen, Hilden, Germany). One µg of total RNA was reverse transcribed into cDNA using an oligo(dT)16 primer. For PCR amplification of Fas, FasL, and ß-actin (control), the following primers were used: (a) Fas, 5'-GCAACACCAAGTGCAAAGAGG-3' and 5'-GTCACTAGTAATGTCCTTGAGG-3'; (b) FasL, 5'-ATGTTTCAGCTCTTCCACCTACAGA-3' and 5'-CCAGAGAGAGCTCAGATACGTTGAC-3'; and (c) ß-actin, 5'-GCCGCCAGCTCACCATGG-3' and 5'-CTCCTCGGGAGCCACACG-3'.

PCR conditions were as follows: an initial 5-min denaturation step was followed by 35 cycles of 30 s of denaturation at 94°C, 30 s of annealing at 58°C, 1 min of primer extension at 72°C, and a terminal primer extension step of 10 min at 72°C. PCR products were visualized after electrophoresis on a 1% agarose gel. 530 melanoma cells and Jurkat cells were examined. Jurkat cells were stimulated with PHA and served as positive control for FasL expression.

Real-time PCR Analysis (TaqMan Assay)
To confirm the PCR results and detect trace amounts of specific mRNA for Fas and FasL, a recently established 5' nuclease assay for detection of PCR products in a real-time mode was used (46) that enables absolute quantification of mRNA copies. A nonextendable oligonucleotide (probe) is labeled with a reporter fluorescent dye (6-carboxy-fluorescein) at the 5' end and with a quencher fluorescent dye (6-carboxy-tetramethyl-rhodamine) at the 3' end. The fluorescence emission after nucleolytic degradation of the probe is measured in real time by using the ABI PRISM 7700 Sequence detection system (TaqMan; ABI Perkin-Elmer, Weiterstadt, Germany). For Fas, the 5' primer (5'-ACTGTGACCCTTGCACCAAAT-3') targets exon 4 with an overlap of two nucleotides to exon 5. Exon 5 is targeted by the probe (5'-AATCATCAAGGAATGCACACTCACCAGC-3'), whereas the 3' primer (5'-GCCACCCCAAGTTAGATCTGG-3') is in exon 6. In the case of FasL, the 5' primer (5'-AAAGTGGCCCATTTAACAGGC-3') has an overlap of two nucleotides in exon 3, where both probe (5'-TCCAACTCAAGGTCCATGCCT-3') and the 3' primer (5'-AAAGCAGGACAATTCCATAGG-3') match. Primers and probes were obtained from ABI Perkin-Elmer. For calibration of the Fas/FasL TaqMan assay, a strand identical RNA standard was generated by using an in vitro T7-polymerase transcription system. After photometric quantification of the synthesized RNA, the absolute number of molecules per volume was calculated to generate a deletion series ranging from 102 to 108. The dilution was supplemented with yeast tRNA as a carrier RNA to compete for binding affinities. Quantification was done by using the TaqMan EZ RT-PCR Kit (ABI Perkin-Elmer). The reaction conditions were 2 min at 50°C, 30 min at 60°C, 5 min at 95°C, and 40–45 cycles with 20 s at 94°C and 1 min at 60°C.

Statistical Analysis
The number of apoptotic cells in FACScan analysis and the fold induction of luciferase activity in in vitro luciferase assays are given as mean ± SD. Student’s t test was used for statistical analysis, and P <= 0.05 was regarded as statistically significant.


    Acknowledgments
 
We thank R. Waterstradt, H. Bergmann, and A. Skrandies for excellent technical assistance.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by DFG Grant 4/1 (to K. H. P.) Back

2 To whom requests for reprints should be addressed, at Department of Dermatology and Venereology, University of Rostock, Augusten Strasse 80, 18055 Rostock, Germany. Phone: 49-381-4949708; Fax: 49-381-4949702; E-mail: manfred.kunz{at}med.uni-rostock.de Back

3 The abbreviations used are: MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; FasL, Fas ligand; FasR, Fas receptor; HIF-1{alpha}, hypoxia-inducible factor 1{alpha}; MAPK, mitogen-activated protein kinase; SAPK, stress-activated protein kinase; JNK, c-Jun NH2-terminal kinase; MBP, myelin basic protein; PHA, phytohemagglutinin; TNF, tumor necrosis factor; GST, glutathione S-transferase; Ab, antibody; moAb, monoclonal antibody; pAb, polyclonal antibody; RT-PCR, reverse transcription-PCR; ß-gal, ß-galactosidase. Back

Received for publication 9/ 5/00. Revision received 1/11/01. Accepted for publication 1/17/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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