Raf/mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)1,2/extracellular signal-regulated kinase1,2 and MKK3,6/p38 mitogen-activated protein kinase pathways play an important role in cellular survival and apoptosis. The results of this study identify novel mechanisms to explain the opposing effects of these pathways in the regulation of apoptosis induction. Our results show that activation of p38 by adenoviral expression of MKK3b or arsenite treatment was followed by rapid dephosphorylation of MEK1,2 and subsequent apoptosis in human skin fibroblasts. Inhibition of p38 activity by SB203580 and adenoviral expression of dominant-negative forms of p38 potently inhibited MEK1,2 dephosphorylation and apoptosis. Strikingly, p38-mediated dephosphorylation of MEK1,2, was not detected in a series of transformed human cell lines. Taken together, we provide evidence for mechanisms unidentified previously that negatively regulates survival signaling during apoptosis induction. In addition, we show that in all transformed cell lines we have studied thus far, the function of this pathway is impaired.

MAPKs3 are a family of eukaryotic serine/threonine protein kinases widely conserved among eukaryotes. MAPKs regulate many cellular processes, such as cell proliferation, migration, differentiation, and death (1). Three MAPK pathways have been characterized in detail: (a) ERK1,2; (b) JNK1,2; and (c) the p38 group of MAPKs (1).

The signaling cascade Raf/MEK1,2/ERK1,2 is prototypically activated by mitogenic growth factors, and it plays a crucial role in the regulation of cell proliferation and survival (1, 2). The critical role of ERK1,2 signaling pathway in cell survival is supported by findings that activated alleles of MEK1 and MEK2 promote cell survival independently of survival factors and that dominant interfering MEK1 and MEK2 alleles disrupt cell survival signaling (2). The role of ERK1,2 pathway in malignancies has been a topic of intensive research lately. Increased activity of this pathway has been detected in several cancers (2, 3, 4, 5). Recently, mutations of B-Raf, which increase activity of MEK1,2-ERK1,2 pathway, were found in >66% of melanomas, and expression of such mutants in NIH3T3 cells led to transformation (3). These studies have indicated that inhibition of this pathway might have important implications in cancer therapy (2, 3, 4, 5, 6). Chemical inhibitors of MEK1,2 are currently being evaluated for treatment of cancer, and it has been shown that they suppress tumor growth and invasion (2, 4, 5).

Pro-apoptotic p38 MAPK pathway is activated by cellular stress, including UV light, arsenite, osmotic shock, and inflammatory cytokines (7). Recent findings indicate a requirement for a correct balance between MEK1,2-ERK1,2 and p38 signaling pathways to ensure appropriate regulation of cell survival (2, 8). However, the molecular mechanisms regulating the balance between ERK1,2 and p38 pathways are largely unknown. We have reported previously that activation of p38 MAPK by arsenite inhibits ERK1,2 signaling pathway and collagenase-1 (MMP-1) promoter activity via PP1/PP2A-dependent dephosphorylation of MEK1,2 (9). Here, we have examined whether p38-mediated inactivation of ERK1,2 signaling cascade affects cell survival. Our results show that p38-mediated dephosphorylation of MEK1,2 mediates initiation of apoptosis by arsenite or p38 activation in normal human skin fibroblasts and rat primary neurons. Importantly, p38-mediated MEK1,2 dephosphorylation was not detected in several cancer cell lines, suggesting that this pathway is suppressed during malignant transformation to promote cell survival.

Cell Cultures.

Normal human skin fibroblast cultures were established from a healthy male volunteer donor (aged 28 years). All transformed cancer cell lines were obtained from American Type Culture Collection. Cells were cultured in DMEM supplement with 10% FCS, 2 mm glutamine, 100 IU/ml penicillin G, and 100 μg/ml streptomycin. Terminally differentiated rat CGNs were a generous gift from Dr. Eleanor Coffey (10).

Reagents and Antibodies.

TPA and sodium m-arsenite were purchased from Sigma Chemical Co. The p38 inhibitor SB203580 and MEK1,2 inhibitor PD98059 were from Calbiochem. Phospho-specific MEK1,2, ERK1,2, JNK, p38, and Akt antibodies and antibodies against total MEK1,2, and p38 were obtained from Cell Signaling Biotechnology. PARP antibody was obtained from Santa Cruz Biotechnology.

Determination of MAPK Activity.

The activation of MEK1,2, p38, JNK, and Akt was determined by Western blotting with antibodies specific for phosphorylated, activated forms of these kinases. Cells were maintained for 18 h in medium supplemented with 1% FCS, treated as indicated, and lyzed in 100 μl of Laemmli sample buffer. Western blotting was performed as described previously (9).

Determination of Cell Viability.

To determine the effect of sodium arsenite on cell viability, cells were seeded in 96-well plates and cultured in 0.5% or 1% FCS/DMEM medium for 24 h. Afterward, medium was changed, and cells were supplied with medium containing sodium arsenite, SB203580, or PD98059 for indicated time periods. The cell viability was determined by CellTiter 96 AQueous nonradioactive cell proliferation assay (Promega).

Detection of Apoptotic Cells.

Human skin fibroblasts were seeded on glass slides and cultured in medium with 1% FCS. Where indicated, cells were infected with recombinant adenovirus for 48 h before seeding on glass slides maintained for different time periods. Afterward, cells were fixed with fresh 4% paraformaldehyde at +4°C for 5 min. Apoptotic cells were detected by TUNEL staining by In Situ Cell Death Detection Kit (Roche). To detect apoptotic cells, nuclei were also stained with Hoechst-33258 (10 μl/ml), analyzed, and photographed by Leica fluorescence microscopy.

Adenoviral Infections of Fibroblasts.

Recombinant replication-deficient adenovirus RAdlacZ (RAd35), empty adenovirus RAd66 (both kindly provided by Gavin W. G. Wilkinson), constitutively active MEK1 (RAd-CA-MKK1-HA; kindly provided by Marco Foschi, University of Florence, Florence, Italy), constitutively active human MKK3b (RAdMKK3bE) and MKK6b (RAdMKK6bE), wild-type p38α (RAdp38α) and p38β (RAdp38β), and dominant-negative p38α [RAdp38α(AF)] and p38β [RAdp38β(AF)] have been described previously (11, 12, 13, 14). Fibroblasts (1.5 × 105) in suspension were infected as described previously with recombinant adenoviruses at a MOI 500 or 1000, which give 100% transduction efficiency (15). After an overnight incubation, the culture medium (DMEM with 1% FCS) was changed, and the cultures were further incubated for indicated time periods. Thereafter, the cell layers were harvested and used for determination of MAPK activation by Western blot analysis with phospho-specific antibodies, cell viability was studied by MTT assay, and apoptotic cells were identified by TUNEL staining, as described above.

Activation of p38 MAPK Is Required for Arsenite-induced Apoptosis and MEK1,2 Dephoshorylation in Fibroblasts.

Our previous observations show that treatment of human skin fibroblasts with arsenite results in p38 MAPK-mediated dephosphorylation of MEK1,2, and inhibition of ERK1,2-mediated gene regulation via p38-mediated activation of protein phosphatases PP1/PP2A (9). To examine the functional consequences of the p38-mediated inhibition of MEK1,2 on cell survival, we treated human skin fibroblasts with increasing concentrations of arsenite and determined cell viability at different time points using the MTT assay. As shown in Fig. 1,A, treatment of fibroblasts with arsenite resulted in reduced cell viability in a dose- and time-dependent manner, the maximal effect noted with 50 μm concentration (Fig. 1 A).

To directly study the role of p38 MAPK activity on arsenite-elicited induction of cell death, human skin fibroblasts were treated with arsenite (50 μm) alone or in combination with increasing concentrations of specific p38 inhibitor, SB203580, and cell viability was monitored using the MTT assay. Supporting a role of p38 in this process, pretreatment of cells with SB203580 dose dependently prevented arsenite-induced cell death (Fig. 1 B). In accordance with the results obtained by the MTT assay, pretreatment of cells with SB203580 at concentrations from 5 to 10 μm also clearly alleviated the arsenite-induced apoptotic phenotype detected by Hoechst staining (data not shown).

To study the regulation of MAPK signaling during stress-induced apoptosis, cells were treated with 50 μm arsenite for different periods of time and activation of MEK1,2, ERK1,2, p38, and JNK was examined by Western blotting using corresponding phospho-specific antibodies. Treatment of cells with arsenite caused rapid activation of p38 MAPK detected after 15 min (Fig. 1,C and data not shown). Interestingly, p38 activation was rapidly followed by potent dephosphorylation of MEK1,2, first detectable at the 30-min time point and inactivation of ERK1,2 at the 1-h time point (Fig. 1,C). Importantly, arsenite-elicited MEK1,2 dephosphorylation was completely blocked by pretreatment of cells with SB203580 (Fig. 1,D). Moreover, activation of JNK did not precede MEK1,2 inactivation, indicating that inhibition of MEK1,2 activity by arsenite is not mediated by the JNK pathway (Fig. 1 C).

Taken together, these results strongly suggest that p38-mediated MEK1,2 dephosphorylation is a prerequisite for arsenite-elicited induction of apoptosis and that protein phosphatase activity induced by arsenite treatment specifically inactivates MEK1,2-ERK1,2 signaling pathway but does not inhibit other studied phosphoproteins: (a) p38; (b) JNK (Fig. 1, C and D); or (c) Akt (data not shown).

Activation of p38 by MKK3b, but not MKK6b, Induces MEK1,2 Dephosphorylation and Promotes Apoptosis.

Recent studies by us and others have provided evidence for specific roles of p38 isoforms in the regulation of different biological processes (12, 15, 16). The experiments described above, in which pretreatment of cells with SB203580 inhibited arsenite-elicited cell death, suggested that the effects of arsenite are mediated by p38α and p38β isoforms because SB203580 only inhibits these isoforms (7). On the basis of reverse transcription-PCR analysis, both p38α and p38β were expressed by human skin fibroblasts used for this study (data not shown). To further elucidate the roles of p38α and p38β in MEK1,2 dephosphorylation, cells were infected with recombinant adenoviruses coding for wild-type or dominant-negative forms of p38α and p38β, and MEK1,2 phosphorylation was assayed after 30-min treatment with arsenite (50 μm). As expected, arsenite treatment of cells infected with a control virus encoding LacZ completely blocked MEK1,2 phosphorylation (Fig. 2,A). Expression of dominant-negative p38α and p38β effectively blocked arsenite-induced dephosphorylation of MEK1,2, as compared with cells overexpressing the wild-type p38α or p38β isoform or LacZ (Fig. 2,A). Furthermore, simultaneous expression of dominant-negative p38α and p38β increased the basal activity of MEK1,2 of cells in the absence of arsenite and completely blocked arsenite-elicited MEK1,2 dephosphorylation (Fig. 2,A). These results indicate that both p38α and p38β mediate arsenite-induced MEK1,2 dephosphorylation and that simultaneous inhibition of both p38 isoforms in normal quiescent cells results in accumulation of phosphorylated MEK1,2. Importantly, adenoviral expression of dominant-negative p38α and p38β also significantly inhibited arsenite-mediated cell death (Fig. 2 B).

Recent studies have shown that in addition to specific roles of p38 protein isoforms, the p38 MAPK kinases, MKK3 and MKK6, also have nonredundant biological effects in vivo(17, 18). Moreover, during the course of this study, it was shown that MKK3 but not MKK6 is required for cytokine withdrawal-induced apoptosis in T cells (19). To investigate the role of MKK3 and MKK6 in apoptosis regulation in human skin fibroblasts, cells were infected with adenoviruses coding for constitutively active forms of MKK3b and/or MKK6b, and appearance of an apoptotic phenotype was first studied by phase-contrast microscopy. Cells infected with RAdMKK3bE showed prominent morphological changes, including cell shrinkage and rounding, whereas cells infected with RAdMKK6bE showed no signs of cell death (Fig. 2,C). To verify that the expression of constitutively active MKK3b induced apoptosis, we performed TUNEL staining of cells 48 h after infection. In accordance with the morphological changes observed, only cultures infected with RAdMKK3bE contained TUNEL-positive cells, whereas no apoptotic cells were detected in cultures infected with RAdMKK6bE or control virus (Fig. 2,D). Importantly, the apoptosis-promoting effect of constitutively active MKK3b was dependent on p38 MAPK activity, because pretreatment of cells with SB203580 abolished the ability of MKK3b to induce apoptosis (Fig. 2 D).

The molecular mechanisms explaining the difference between MKK3 and MKK6 in apoptosis regulation are still not known. On the basis of the results of this work, one plausible mechanism for MKK3b to exert a pro-apoptotic effect might be its capability to induce MEK1,2 dephosphorylation. To test this presumption, human skin fibroblasts were infected with MKK3b and MKK6b adenoviruses and analyzed by Western blotting using a phosphospecific MEK1,2 and p38 antibody. In support of our hypothesis, activation of endogenous p38 proteins by expression of constitutively active MKK3b resulted in dephosphorylation of MEK1,2 to a level comparable with that caused by treatment of cells with arsenite, whereas overexpression of MKK6b had no effect on MEK1,2 phosphorylation (Fig. 2 E). Importantly, MKK3b overexpression-induced MEK1,2 dephosphorylation was blocked by pretreatment of cells with SB203580 (data not shown).

Finally, to elucidate the importance of MEK1,2 activity for survival signaling in these cells, we inhibited the activity of MEK1,2 with a specific chemical inhibitor, PD98059, for 48 h and subsequently studied the appearance of apoptotic cells by TUNEL staining. As shown in Fig. 2,D, inhibition of MEK1,2 by PD98059 in cells infected with the empty control virus resulted in the induction of apoptosis similar to expression of constitutively active MKK3b, demonstrating that inhibition of MEK1,2 activity promotes apoptosis in these cells. The survival-promoting role of MEK1 is also supported by the results showing that adenoviral expression of constitutively active form of MEK1 could rescue cells from arsenite-induced apoptosis (Fig. 2,B). Interestingly, adenoviral expression of MKK6b protected cells from apoptosis initiation, caused by removal of ERK1,2-mediated survival signaling by PD98059 treatment (Fig. 2 D). The survival-promoting role of MKK6 is in accordance with results showing that activity of MKK6 is required for γ-irradiation-induced G(2) arrest and cell survival (20).

Transformed Cell Lines Are Resistant to Arsenite-dependent MEK1,2 Dephosphorylation.

The results above indicate that the cellular stress response mediated by the MKK3b-p38 pathway induces dephosphorylation of MEK1,2 and subsequent apoptosis in human skin fibroblasts. To investigate cell specificity of this response, we next asked whether arsenite-elicited p38 activation results in MEK1,2 dephosphorylation in another primary cell line. To this end, CGNs from rat brain were terminally differentiated in culture and MEK1,2 phosphorylation after treatment with TPA, and arsenite treatments were evaluated by Western blotting. TPA treatment was used to allow reliable detection of phosphorylated MEK1,2. In accordance with the results obtained with dermal fibroblasts, treatment of CGN cells with arsenite resulted in rapid and potent activation of p38, which was accompanied by potent suppression of TPA-induced MEK1,2 phosphorylation at 30-min and 1-h time points (Fig. 3, A and B).

For a cancer cell to survive under stress conditions, it would be beneficial to suppress p38-mediated MEK1,2 dephosphorylation, thereby preventing apoptosis induction. In this regard, we sought to study if stress-activated MEK1,2 dephosphorylation occurs in transformed human cell lines. To this end, the effect of arsenite treatment on MEK1,2 phosphorylation was studied as described above in a panel of transformed human cell lines. The cell lines used for this experiment represent cells from different histogenetic origins and causes of transformation (Table 1). Importantly, all cell lines studied were responsive to arsenite treatment as assessed by Western blot analysis using the phospho-specific p38 antibody (data not shown). Strikingly, arsenite treatment did not induce dephosphorylation of MEK1,2 at 30-min or 1-h time points in any of the transformed cell lines studied (Table 1). A representative experiment done in HT-1080 human fibrosarcoma and HeLa cells is shown in Fig. 4, A and B. To strengthen our hypothesis that MEK1,2 activity is important for survival of these cancer cells, we next treated these cells with PD98059 and studied cell viability after 24 h by MTT assay. Results shown in Fig. 3,C demonstrate that, with an exception of K562 cells, all cell lines studied display decreased cell viability on PD98059 treatment. The reason for differential response in K562 cells is not yet known. To confirm that inhibition of MEK1,2 activity in these cells causes apoptosis, we show that treatment of HT-1080 cells with PD98059 results in proteolytical cleavage of PARP protein, considered as a specific sign of apoptosis induction (Fig. 3 D). Taken together, these novel results indicate that p38-mediated MEK1,2 dephosphorylation is involved in the cellular stress response of normal human skin fibroblasts and rat primary granular neurons, but this response is impaired in all of the transformed cells we have studied.

We do not have information on the mechanism by which transformed cells inhibit this response as yet. However, these results reveal a strategy unidentified previously that allow transformed cells to escape death by suppressing inhibition of survival signaling under stress conditions.

Further understanding of molecular mechanisms how the activity of ERK1,2 pathway is regulated might prove to be beneficial for development of cancer therapies. In this study, we have identified a novel pathway used by nontransformed cells to inhibit MEK1,2 activity and cell survival. We show that the pro-apoptotic effect of the p38 MAPK pathway relies on dephosphorylation of MEK1,2. In support of this model, we show that inhibition of MEK1,2 activity by p38-mediated dephosphorylation or with the inhibitor PD98059 abolishes the cell survival-promoting effect of ERK1,2 pathway.

In all transformed cell lines we have studied thus far, MEK1,2 dephosphorylation does not occur in response to p38 activation. As we showed previously, arsenite-elicited MEK1,2 dephosphorylation is mediated by PP1/PP2A protein phosphatases (9). Recent studies have indicated that inhibition of PP2A activity is a prerequisite for transformation of primary human cells (21, 22, 23, 24). Taking into consideration the role of PP2A as a negative regulator of MEK1,2 (21, 24, 25), and the requirement of MEK1,2 activity for cancer cell survival (2, 4, 5), we propose that p38/PP2A-mediated MEK1,2 dephosphorylation is inhibited in transformed cell lines as a means of suppressing apoptosis. We have already shown that p38 mediates induction of invasion proteinases and malignant cell invasion (26). It is, therefore, reasonable that p38-mediated inhibition of MEK1,2 has been uncoupled in malignant cells without compromising the p38 activity that is important for invasion.

Interestingly, it was recently shown that cellular transformation by a constitutively activated Raf-1 allele does not occur without simultaneous suppression of p38 activity (27). Our data suggest the presence of a continuous negative feedback from p38α and p38β to MEK1,2 as simultaneous inhibition of p38α and p38β isoforms in normal quiescent cells resulted in accumulation of phosphorylated MEK1,2 (Fig. 2 A). This negative regulation of MEK1,2 in normal cells could be considered a means to control MEK1,2-mediated proliferation and expression of transformation-related genes. In this regard, our findings provide a plausible mechanistic explanation for the requirement of the suppression of p38 activity during cellular transformation. It is also tempting to speculate that components of MKK3-p38α/β-PP2A pathway might function as tumor suppressors.

Our results, regarding the nonredundant roles of MKK3b and MKK6b in the regulation of cell survival, are in good agreement with targeted gene disruption studies from Mkk3−/− and Mkk6−/− mice (17, 18, 19). We show for the first time that the apoptosis-promoting activity of MKK3b correlates with its capacity to induce MEK1,2 dephosphorylation, thus providing new insights of the molecular mechanism by which MKK3 and MKK6 differentially regulate cell fate.

In conclusion, the results presented in this study identify a novel pathway that negatively regulates survival signaling during apoptosis induction. In addition, we show that in all transformed cell lines we have studied thus far, the function of this pathway is impaired. It will be of great importance to further study the relevance of these findings in respect to apoptosis resistance of cancer cells and whether restoration of MKK3-p38-PP2A-MEK1,2 signaling pathway represents a novel approach for sensitizing cancer cells to chemotherapy.

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 grants from the Foundation of Finnish Cancer Institute, the Academy of Finland (Projects 30985, 45996, and 878179), Sigrid Juselius Foundation, Cancer Research Foundation of Finland, Turku University Hospital (EVO Grant 13336), and a research contract from the Finnish Life and Pension Insurance Companies.

3

The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; TPA, 12-O-tetradecanoyl-13-phorbol acetate; JNK, c-Jun NH2-terminal protein kinase; MKK, ; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; MOI, multiplicity of infection; PARP, poly(ADP-ribose) polymerase; CGN, cerebellar granular neuron; MTT<3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Fig. 1.

Activation of p38 MAPK is required for arsenite-induced apoptosis and MEK1,2 dephosphorylation in human skin fibroblasts. In A, serum-starved human skin fibroblasts were treated with arsenite (30, 50, 80, and 100 μm) for indicated periods of time, after which, the cell viability was measured by MTT assay. Mean ± SE of values from three independent experiments each performed with four parallel cultures are shown. In B, serum-starved human skin fibroblasts were treated with arsenite (50 μm) for indicated time periods and assayed for cell viability by MTT assay. Where indicated, cells were pretreated with specific inhibitor of p38 activity SB203580 (5, 10, and 20 μm) for 1 h before the addition of arsenite. Thereafter, fresh SB203580 was added every 24 h until the end of the experiment. Mean ± SE of values from three independent experiments each performed with four parallel cultures are shown. In C, serum-starved human skin fibroblasts were treated with arsenite (50 μm) for indicated periods of time, and cell lysates were subjected to Western blot analysis using antibodies specific for phosphorylated forms of MEK1,2, ERK1,2, JNK, and p38. Equal loading was confirmed by antibodies against total p38 and MEK1,2. In D, serum-starved human skin fibroblasts were treated with arsenite (50 μm) for 30 min. Where indicated, cells were pretreated with SB203580 (20 μm) for 1 h before the addition of arsenite. Cell lysates were subjected to Western blot analysis using antibodies specific for the phosphorylated form of MEK1,2. Equal loading was confirmed by antibody against total MEK1,2.

Fig. 1.

Activation of p38 MAPK is required for arsenite-induced apoptosis and MEK1,2 dephosphorylation in human skin fibroblasts. In A, serum-starved human skin fibroblasts were treated with arsenite (30, 50, 80, and 100 μm) for indicated periods of time, after which, the cell viability was measured by MTT assay. Mean ± SE of values from three independent experiments each performed with four parallel cultures are shown. In B, serum-starved human skin fibroblasts were treated with arsenite (50 μm) for indicated time periods and assayed for cell viability by MTT assay. Where indicated, cells were pretreated with specific inhibitor of p38 activity SB203580 (5, 10, and 20 μm) for 1 h before the addition of arsenite. Thereafter, fresh SB203580 was added every 24 h until the end of the experiment. Mean ± SE of values from three independent experiments each performed with four parallel cultures are shown. In C, serum-starved human skin fibroblasts were treated with arsenite (50 μm) for indicated periods of time, and cell lysates were subjected to Western blot analysis using antibodies specific for phosphorylated forms of MEK1,2, ERK1,2, JNK, and p38. Equal loading was confirmed by antibodies against total p38 and MEK1,2. In D, serum-starved human skin fibroblasts were treated with arsenite (50 μm) for 30 min. Where indicated, cells were pretreated with SB203580 (20 μm) for 1 h before the addition of arsenite. Cell lysates were subjected to Western blot analysis using antibodies specific for the phosphorylated form of MEK1,2. Equal loading was confirmed by antibody against total MEK1,2.

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Fig. 2.

Activation of p38 by MKK3b-p38α/β pathway inhibits MEK1,2 activity and promotes apoptosis in human skin fibroblasts. In A, human skin fibroblasts were transduced with control adenovirus RAdlacZ and adenoviruses harboring Flag-tagged, wild-type p38α and p38β and dominant-negative p38α [p38α(AF)] and p38β [p38β(AF)] and at MOI 500 for 48 h. At the end of the incubation period, the cells were treated with arsenite (50 μm) for 30 min, and cell lysates were subjected to Western blot analysis. Equal loading and expression of p38 isoforms were detected by antibodies against total MEK1,2 and Flag. In B, human skin fibroblasts were transduced with control adenovirus RAdlacZ and adenoviruses harboring dominant-negative p38α [p38α(AF)] and p38β [p38β(AF)] or with constitutively active forms of MEK1 (MEK1ca) for 24 h at MOI 500. Cultures were then treated with arsenite (50 μm) for 48 h and assayed for cell viability by MTT assay. Mean ± SE cell viability compared with untreated control cultures from three independent experiments each performed with four parallel samples are shown. Statistical significance determined by Student’s t test as comparing with the RAdlacZ control: ∗∗, P < 0.001. In C, human skin fibroblasts were infected with RAdMKK3bE, RAdMKK6bE, and empty control virus (RAd66) at MOI 500 for 72 h. Thereafter, cell morphology was studied by phase-contrast microscopy (magnification, ×20). In D, human skin fibroblasts were infected with RAdMKK3bE, RAdMKK6bE, and control virus (RAd66) at MOI 1000 for 48 h. Where indicated, cells were treated with SB203580 (20 μm) and PD98059 (40 μm) added 3 h after infection and again every 24 h until the end of the experiment. Thereafter, apoptotic cells were detected by TUNEL staining, and cultures were examined by fluorescence microscopy (magnification, ×20). In E, human skin fibroblasts were infected with recombinant adenoviruses coding for constitutively active MKK3b (MKK3bE), MKK6b (MKK6bE), and control virus RAdlacZ at MOI 500 and incubated for 48 h. At the end of the incubation period, arsenite (50 μm) was added for 30 min. Thereafter, cell lysates were analyzed for activation of MEK1,2 and p38 by Western blot analysis using phospho-specific antibodies.

Fig. 2.

Activation of p38 by MKK3b-p38α/β pathway inhibits MEK1,2 activity and promotes apoptosis in human skin fibroblasts. In A, human skin fibroblasts were transduced with control adenovirus RAdlacZ and adenoviruses harboring Flag-tagged, wild-type p38α and p38β and dominant-negative p38α [p38α(AF)] and p38β [p38β(AF)] and at MOI 500 for 48 h. At the end of the incubation period, the cells were treated with arsenite (50 μm) for 30 min, and cell lysates were subjected to Western blot analysis. Equal loading and expression of p38 isoforms were detected by antibodies against total MEK1,2 and Flag. In B, human skin fibroblasts were transduced with control adenovirus RAdlacZ and adenoviruses harboring dominant-negative p38α [p38α(AF)] and p38β [p38β(AF)] or with constitutively active forms of MEK1 (MEK1ca) for 24 h at MOI 500. Cultures were then treated with arsenite (50 μm) for 48 h and assayed for cell viability by MTT assay. Mean ± SE cell viability compared with untreated control cultures from three independent experiments each performed with four parallel samples are shown. Statistical significance determined by Student’s t test as comparing with the RAdlacZ control: ∗∗, P < 0.001. In C, human skin fibroblasts were infected with RAdMKK3bE, RAdMKK6bE, and empty control virus (RAd66) at MOI 500 for 72 h. Thereafter, cell morphology was studied by phase-contrast microscopy (magnification, ×20). In D, human skin fibroblasts were infected with RAdMKK3bE, RAdMKK6bE, and control virus (RAd66) at MOI 1000 for 48 h. Where indicated, cells were treated with SB203580 (20 μm) and PD98059 (40 μm) added 3 h after infection and again every 24 h until the end of the experiment. Thereafter, apoptotic cells were detected by TUNEL staining, and cultures were examined by fluorescence microscopy (magnification, ×20). In E, human skin fibroblasts were infected with recombinant adenoviruses coding for constitutively active MKK3b (MKK3bE), MKK6b (MKK6bE), and control virus RAdlacZ at MOI 500 and incubated for 48 h. At the end of the incubation period, arsenite (50 μm) was added for 30 min. Thereafter, cell lysates were analyzed for activation of MEK1,2 and p38 by Western blot analysis using phospho-specific antibodies.

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Fig. 3.

Transformed cell lines require MEK1,2 activity for their survival and are resistant to arsenite-induced MEK1,2 dephosphorylation. In A, serum-starved cell lines were incubated with TPA (60 ng/ml) alone or in combination with arsenite (80 μm) for 30 min or 1 h as indicated. Thereafter, cells were lysed, and total protein extracts were subjected to Western blot analysis to detect p38 and MEK1,2 activation by using phospho-specific p38 and MEK1,2 antibodies. B, the quantitation of A by MCID analysis. In C, indicated cell lines were serum starved and subsequently treated with PD98059 for 24 h, after which, the cell viability was measured by MTT assay. Mean ± SE of value from a representative experiment performed with four parallel cultures are shown. Experiment was repeated three times with equal results. In D, HT-1080 cells were serum starved and subsequently treated with PD98059 for 24 h. Induction of apoptosis was thereafter studied by Western blotting of PARP protein. ∗, proteolytically processed form of PARP.

Fig. 3.

Transformed cell lines require MEK1,2 activity for their survival and are resistant to arsenite-induced MEK1,2 dephosphorylation. In A, serum-starved cell lines were incubated with TPA (60 ng/ml) alone or in combination with arsenite (80 μm) for 30 min or 1 h as indicated. Thereafter, cells were lysed, and total protein extracts were subjected to Western blot analysis to detect p38 and MEK1,2 activation by using phospho-specific p38 and MEK1,2 antibodies. B, the quantitation of A by MCID analysis. In C, indicated cell lines were serum starved and subsequently treated with PD98059 for 24 h, after which, the cell viability was measured by MTT assay. Mean ± SE of value from a representative experiment performed with four parallel cultures are shown. Experiment was repeated three times with equal results. In D, HT-1080 cells were serum starved and subsequently treated with PD98059 for 24 h. Induction of apoptosis was thereafter studied by Western blotting of PARP protein. ∗, proteolytically processed form of PARP.

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Table 1

Dephosphorylation of MEK1,2 in response to arsenite treatment

Cell lineResponsivenessaMorphologyTissueTumorigenic
HSF Fibroblast Skin No 
CGN Neuron Cerebellum No 
HK293 − Epithelial Kidney; adenovirus 5 transformed Yes 
HT-1080 − Epithelial Fibrosarcoma Yes 
WM 266-4 − Epithelial Skin; melanoma Yes 
A2058 − Epithelial Skin; melanoma Yes 
HeLa − Epithelial Cervix; adenocarcinoma N/A 
Jurkat − Lymphocyte Acute T-cell leukemia N/A 
K562 − Lymphocyte Chronic mylogenous leukemia N/A 
Cell lineResponsivenessaMorphologyTissueTumorigenic
HSF Fibroblast Skin No 
CGN Neuron Cerebellum No 
HK293 − Epithelial Kidney; adenovirus 5 transformed Yes 
HT-1080 − Epithelial Fibrosarcoma Yes 
WM 266-4 − Epithelial Skin; melanoma Yes 
A2058 − Epithelial Skin; melanoma Yes 
HeLa − Epithelial Cervix; adenocarcinoma N/A 
Jurkat − Lymphocyte Acute T-cell leukemia N/A 
K562 − Lymphocyte Chronic mylogenous leukemia N/A 
a

Responsiveness was determined by comparing the level of MEK phosphorylation at 30-min and 1-h time points detected by Western analysis between serum-starved cells treated with TPA (60 ng/ml) alone or in combination with arsenite (80 μm).

We thank Sari Pitkänen and Marjo Hakkarainen for skillful technical assistance. We also thank Dr. Eleanor Coffey and Vesa Hongisto for providing us differentiated CGNs. Finally, we thank Drs. Dirk Bohmann and Panu Jaakkola for critical comments on this manuscript.

1
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