Carcinogenesis

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Carcinogenesis, Vol. 23, No. 11, 1831-1838, November 2002
© 2002 Oxford University Press

CANCER BIOLOGY

The role of MAPK pathways in the action of chemotherapeutic drugs

Simone Boldt¹, Ulrich H. Weidle² and Walter Kolch^1,3,4

¹ The Beatson Institute for Cancer Research, Cancer Research UK, Bearsden, Glasgow G61 1BD, UK,
² Roche Diagnostics GmbH, Pharma Research, Penzberg, Germany and
³ Institute for Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK

	Abstract

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In this study we have investigated the role of mitogen-induced and stress-activated MAP kinase pathways in the cellular response to taxol, etoposide and ceramide in three different human cancer cell lines: HeLa cervical carcinoma, MCF7 breast cancer and A431 squamous carcinoma cells. The mitogen-induced ERK MAPKs were linked to cell proliferation and survival, whereas the stress-activated MAPKs, p38 and JNK, were connected with apoptosis. Our results show that all drugs activated MAPKs, but that the extent and kinetics of activation were different. In order to assay the biological consequences of drug-induced MAPK activation we employed selective MAPK inhibitors and measured both long-term clonogenic survival as well as short-term parameters including apoptosis, mitochondrial metabolic integrity and cell cycle progression. Our results show that drug induced toxicity is not correlated with any singular parameter, but rather a combination of effects on cell cycle and apoptosis. In certain constellations the modulation of MAPK pathways could enhance or decrease drug efficacies. These effects mainly pertained to the regulation of apoptosis and clonogenic survival, but they were highly dependent on the combination of drug and cell line without any clear patterns of correlations emerging. These results suggest that the modulation of MAPK pathways to enhance the efficacy of chemotherapeutic drugs is of limited value unless it is tailored to the specific combination of drug and cancer.

	Introduction

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Many of the commonly used chemotherapeutic drugs exhibit some selectivity for tumour cells, but it is largely unknown why and exactly how they induce tumour cell death. In recent years it has emerged that they impinge on cellular signalling pathways and often may recruit them to induce apoptosis in cancer cells (1–3). Apoptosis seems to be one of the major safeguards against uncontrolled proliferation. Nature has linked these two diametrically opposed biological outcomes to ensure that multicellular organisms can cope with the onslaught of a highly mutagenic environment that generates millions of potential cancer cells every day (4,5). The Myc proto-oncogene is a classical paradigm (6,7). Indispensable for cell cycle progression on the one hand, Myc efficiently induces apoptosis, if proliferation is not licensed by appropriate external cues. Because the intimate relationship between proliferation and apoptosis cannot be separated, cancer cells are often hypersensitive to apoptosis induction. They often only can avoid apoptosis because they have lost or can bypass check points that control cell cycle progression (8).

Most chemotherapeutic drugs induce cellular damage on such a massive scale that they can engage one or more of these check points to drive cancer cells into apoptosis (9). Obviously, chemotherapy could be enhanced if the efficiency of recruiting these check points or the stringency of executing them could be increased. Such measures potentially could sharply improve the selectivity of chemotherapeutic drugs, because they utilize physiological mechanisms that should be fairly innocuous to normal cells. Good candidates for such modulators are MAPK pathways (10). These signalling modules consist of a three-tiered kinase core where a MAPKKK activates a MAPKK that activates a MAPK (11,12). The primordial MAPK cascades are ubiquitously expressed and respond to a wide variety of external cues and drugs. The ERK module is activated by mitogenic stimuli and is thought to mediate cell proliferation and survival. The JNK and p38 MAPK modules are activated in response to cellular stress and seem to be exert both protective as well as pro-apoptotic functions.

In order to investigate the effects of chemotherapeutic drugs on apoptosis and MAPK pathway activation we used a model system consisting of three human cancer cell lines and three compounds. A431 is a squamous carcinoma cell line that over expresses the EGF receptor (13) and due to this fact features a constitutive activation of receptor dependent signalling processes. HeLa is derived from a cervical carcinoma, which is infected with HSV and expresses E6 and E7, viral proteins that neutralize the Rb and p53 tumour suppressor proteins (14,15). MCF7 is an estrogen responsive mammary carcinoma cell line that over expresses the anti-apoptotic Bcl-2 protein (16). These cell lines are widely used to study drug responses, because they represent common human cancers and feature different transforming principles important to the aetiology of cancer. The following compounds were used:

1. Taxol, an agent that targets microtubules and is one of the few chemotherapeutic drugs effective against a wide range of solid tumours (17).
   
2. Etoposide, a well-established chemotherapeutic drug that induces DNA damage by inhibiting topoisomerase II (18).
   
3. Ceramide, which is a synthetic lipid that has not found therapeutical application, but is a potent apoptosis inducing substance that has been described as a second messenger of TNF and other apoptosis inducing stimuli (19).
   

	Materials and methods

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Cell lines

• A431: human squamous carcinoma
  
• HeLa: human cervix carcinoma
  
• MCF7: human breast epithelial carcinoma.
  

A431 and HeLa cells were maintained in DMEM (Life Technologies, Paisley, UK) supplemented with 10% foetal bovine serum (Harlan Sera-Lab, Loughborough, UK). MCF7 cells were maintained in RPMI (Life Technologies) with 10% FBS.

Antibodies
Phosphorylation specific antibodies to JNK, c-Jun and p38 were from Cell Signaling, Herts, UK. Anti-phosphorylated MAP kinase was purchased from Sigma, Gillingham, UK. Anti-Bcl-2 monoclonal antibody was from Roche, Lewes, UK, anti-cleaved PARP antibody was from Promega, Southampton, UK, and anti-MAPKAP kinase 2 was from Upstate, Milton Keynes, UK. All other antibodies were from Santa Cruz, Wiltshire, UK.

Clonogenic assay
Clonogenic assays were carried out on growing cells. Before the induction of apoptosis cells were pre-incubated for 0.5–1 h with 40 µM PD98059, 5 µM SB203580 or, as a control, DMSO to a final concentration of 0.2%. After treatment with taxol, etoposide or ceramide, cells were washed twice with PBS and incubated in normal growth medium in the presence of inhibitors or DMSO for a further 6–20 h. After this, the cells were trypsinized and seeded in 1:20, 1:60 and 1:200 dilutions in normal growth medium. Colonies formed within 1–2 weeks. The cells were then fixed in 50% methanol in PBS followed by 100% methanol for 10 min each and stained with Giemsa (BDH Laboratory Supplies, Dorset, UK). Assays were analysed densitometrically using TINA2.07d (raytest Isotopenmessgeraete GmbH, Straubenhandt, Germany).

Western blotting
For SDS–PAGE, cells were washed twice with ice-cold PBS and lysed in 20 mM Tris–HCl (pH 8.0), 140 mM NaCl, 1% Triton X-100, 0.05% SDS, 10 mM NaF, 10 mM ß-glycerophosphate, 0.5 mM Na₃VO₄, 1 mM PMSF, 10 µg/ml aprotinin and 10 µg/ml leupeptin. After centrifugation for 10 min at 21000 g the protein content of the lysates was determined using a BCA assay kit (Pierce, Rockload, IL, US). Equalized lysates were mixed with protein sample buffer and gel electrophoresis and blotting were carried out according to standard protocols. Blots were quantified using TotalLab software.

JNK kinase assay
JNK activity was assayed using a SAPK/JNK Kinase Assay Kit (Cell Signaling, Herts, UK) following the manufacturer's instructions. Alternatively, GST-cJun (1–70) fusion protein beads were produced from E.coli according to standard protocols. c-Jun phosphorylation was monitored using the phospho-c-Jun (Ser63) antibody from the Assay Kit.

MAPKAP kinase 2 assay
To monitor MAPKAP kinase 2 activity, cells were lysed in JNK lysis buffer. After determination of the protein content, MAPKAP kinase 2 was precipitated using 2 µg/mg anti-MAPKAP kinase 2 antibody and 15 ml protein G agarose (Roche, Lewes, UK) for 4 h or overnight at 4°C. Precipitates were washed three times in a buffer containing 20 mM Hepes, 50 mM NaCl, 2.5 mM MgCl₂, 0.1 mM EDTA and 0.05% Triton X100. Kinase reactions were carried out for 30 min at 30°C in 20 mM Hepes, 20 mM MgCl₂, 10 mM ß-Glycerophosphate, 0.5 mM Na₃VO₄, 2 mM DTT, 10 mM ATP supplemented with 10 µCi [gamma-³²P]ATP and 5 µg HSP25 (Sigma, Gillingham, UK) per reaction. Reactions were stopped by the addition of protein sample buffer and western blots were analysed by autoradiography.

Cell cycle analysis
For cell cycle analysis, adherent cells from one 35 mm dish were harvested by trypsinization and combined with detached cells from the same sample. Cells were washed once in ice-cold PBS and resuspended in 100 µl PBS. After the addition of 900 µl 70% ice-cold ethanol cells were incubated on ice for 1 h or overnight. Cells were then sedimented by centrifugation for 5 min at 400 g and incubated in 300 µl PBS containing 250 µg/ml RNase A and 10 µg/ml propidium iodine for 30 min prior to FACS analysis. Data were analysed using ModFit LT software.

Cell viability assay
For cell viability assays, cells were seeded in 96-well plates at a density of 1000 cells per well. Cells were pre-incubated for 0.5–1 h with 40 µM PD98059, 5 µM SB203580 or, as a control, DMSO to a final concentration of 0.2% and then treated with 50 nM taxol, 40 µg/ml etoposide or 20 µM ceramide overnight. Cells were then washed twice in PBS and incubated in normal growth medium for 6 h before cell viability assays were carried out using Cell Proliferation Reagent WST-1 (Roche, Lewes, UK) according to the manufacturer's instructions.

	Results

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Pharmacological induction of apoptosis in human cancer cell lines
A431, MCF7 and HeLa cells were treated with different drugs and apoptosis induction was monitored by examining PARP cleavage as surrogate marker. The appearance of the 85 kDa proteolytic fragment of PARP is indicative of caspase activation and apoptosis induction (20). As Figure 1A shows, PARP cleavage was readily apparent in response to growth factor withdrawal (serum starvation) and treatment with clinically relevant doses of taxol or etoposide (21–23). Overnight treatment with 20 µM ceramide was also sufficient to induce apoptosis in these cell lines. Although MCF7 cells were sensitive to these agents (Figures 4 and 6), they did not display PARP cleavage. The reason for this is probably related to the lack of detectable caspase 3 expression (Figure 1B) (24). Also, the MCF7 cells used in this study displayed a robust over expression of Bcl-2 (Figure 1B), which can counteract the activation of the caspase cascade via the mitochondrial route (25).

View larger version (46K): [in this window] [in a new window]   Fig. 1. Drug sensitivity of different human cancer cell lines. (A) Induction of PARP cleavage. Growing cells were treated overnight with 50 nM taxol, 40 µg/ml etoposide, 20 µM C6-ceramide or serum starved for the same period of time. Cell lysates analysed for the 85 kDa fragment of cleaved PARP by immunoblotting. (B) Expression of Bcl-2 and Procaspase-3. Growing cells were lysed and equal amounts of protein analysed for expression of the indicated proteins by immunoblotting.

 

View larger version (31K): [in this window] [in a new window]   Fig. 4. The effect of ERK and p38 inhibitors on the drug induced reduction of mitochondrial metabolic function. Cells were treated and analysed using the WST assay kit as described in Materials and methods. Samples were analysed in triplicates and the error bars indicate the standard error of the triplicate samples. Data shown are representative of two or three independent experiments.

 

View larger version (99K): [in this window] [in a new window]   Fig. 6. The effect of ERK and p38 inhibitors on drug induced reduction of clonogenic survival. Cells were pre-treated with the inhibitors or DMSO as a control as described in Materials and methods. Apoptosis was stimulated with 50 nM taxol for 3 h or 40 µg/ml etoposide for 1 h in all cell lines. A431 cells were treated with 20 µM C6-ceramide for 6 h, HeLa and MCF7 were treated overnight with the same concentration. The right-hand panel shows the quantification of the left-hand panel. Data shown are representative of four to five independent experiments.

 
Activation of MAPK cascades by apoptosis inducing drugs
Ceramide, taxol and etoposide have very different intracellular targets, yet all are able to induce apoptosis. The underlying mechanism(s) is (are) incompletely known, and it is unclear where they converge on the apoptotic machinery. Plausible candidates are MAPK pathways which have been implicated as mediators of apoptosis in response to a variety of external stimuli (10). The first descriptions of their role pinpointed the ERK pathway as protective and JNK as pro-apoptotic (26). In addition, it has been shown that the strength and duration of signalling is crucial for the biological outcome (27).

Therefore, we tested the effects of these agents on ERK, JNK and p38 activity over an extended time course (Figure 2). Because HeLa cells are very sensitive to apoptosis induced by taxol and ceramide we could monitor MAP kinase activation in response to these agents for only 24 and 18 h respectively, compared with 36 h for the other cell lines and etoposide treated HeLa cells. Treatment with the drugs had no consequences on the expression levels of ERK, JNK and p38 (data not shown). Phosphospecific antibodies were used to selectively stain the activated form of ERK (Figure 2A). Interestingly, the effects of the drugs on ERK and JNK activities were highly divergent, but mainly depending on the cell type rather than the type of drug. In A431 cells the ERK pathway is constitutively hyper-activated due to the over expression of the EGF receptor. Nevertheless all drugs enhanced ERK activation with delayed, but sustained kinetics. ERK activation by all agents was biphasic with a rapid growth factor like induction after 15 min. The activity returned to almost baseline between 6 and 9 h, and then rose again at 12 h. JNK activation could not be detected using the available phosphospecific antibodies and had to be monitored by kinase assays. JNK activity was low, but also was induced by all agents (Figure 2B). A biphasic activation pattern similar to that of ERK was seen for JNK activity in response to ceramide or etoposide. In contrast, taxol induced a slow, but steady increase of JNK activity. In MCF7 cells the activities of ERK and JNK exhibited only little response to drug treatment compared to A431 and HeLa cells. Only ceramide produced a clear biphasic ERK activation similar to that seen in HeLa. Taxol induced no initial activation of ERK, but a transient repression between 9 and 12 h, which was followed by a slight hyper activation. Taxol also induced a small biphasic JNK activation, whereas the other drugs caused a transient repression followed by a recovery of activity between 6 and 18 h and another decline thereafter.

View larger version (54K): [in this window] [in a new window]   Fig. 2. Drug-induced activation of MAPK pathways. (A) Time course of ERK activation. Growing cells were treated with 50 nM taxol, 40 µg/ml etoposide or 20 µM C6-ceramide in normal growth medium for the indicated time (in hours). Cells were lysed in JNK assay lysis buffer. Samples were normalized to equal protein contents and used to determine ERK activation by immunoblotting with phospho-ERK (P-ERK) specific antibodies. Data shown are representative of five independent experiments. (B) Time course of JNK activity. Samples were prepared as in (A) and JNK activity assayed by the phosphorylation of GST-Jun as described in Materials and methods. Data shown are a representative of five independent experiments. (C) Time course of MAPKAP-2 activity. Cells were treated as in (A) and MAPKAP-2 activity was assayed. Shown is a typical example of three independent experiments. The activity of the respective kinases relative to the controls is indicated in the figure.

 
As with JNK, the activity profile of p38 could not be traced with the available phosphospecific antibodies, because the changes in activity were below the threshold of detection. Therefore, the activity of MAPKAP-2, a direct p38 substrate (28), was assayed instead (Figure 2C). Only etoposide induced MAPKAP-2 activation in all three cell lines. In order to verify that MAPKAP-2 activity reflects p38 activity, the p38 inhibitor SB203580 was employed. SB203580 inhibited both anisomycin (a bona fide chemical inducer of p38 kinase activity) and etoposide induced MAPKAP-2 activation in a dose dependent manner validating the use of MAPKAP-2 as a surrogate for measuring p38 activation (data not shown).

Relationships between MAPK activation and biological drug effects
All drugs induced the activation of different MAPK pathways in a cell type specific manner. Chemotherapeutic drugs affect different cellular functions including cell cycle, cell viability and survival. In order to dissect whether MAPK activation is causal to any of these actions of chemotherapeutics we employed specific inhibitors of MEK and p38.

First, we investigated whether the inhibition of MEK or p38 affects apoptosis induction (Figure 3). As a marker for apoptosis PARP cleavage was used (20). In A431 cells the inhibitors had only small effects on PARP cleavage, although they efficiently inhibited the respective kinases (data not shown). However, in HeLa cells the inhibition of p38 suppressed PARP cleavage, whereas the inhibition of ERK increased it. The latter observation supports the original reports that the ERK pathway prevents apoptosis while p38 promotes it (26). However, the results also indicate that this phenomenon is cell type specific and cannot explain the action of the drugs in general.

View larger version (33K): [in this window] [in a new window]   Fig. 3. The effect of ERK and p38 inhibitors on drug induced apoptosis. A431 and HeLa cells were pre-treated for 1 h with 40 µM PD98059, 5 µM SB203580 or DMSO to a final concentration of 0.2%. The cells were then incubated overnight in the presence of the inhibitors together with 50 nM taxol, 40 µg/ml etoposide or 20 µM C6-ceramide. Lysates were equalized for protein content and PARP cleavage was analysed by immunoblotting. The amount of cleaved PARP relative to cells treated with no drug or inhibitor is indicated in the figure. The samples were analysed on the same blot and the equality of loading was verified by immunoblot with anti-ERK (not shown). Data shown are a representative of four experiments.

 
In parallel, cell viability was scored using the WST assay which measures the metabolic function of the mitochondria. Such assays are widely used to assay cell proliferation and survival. All drugs reduced mitochondrial metabolism (Figure 4). Surprisingly, neither the inhibition of MEK nor p38 exerted profound effects. In HeLa cells p38 inhibition slightly enhanced viability in response to etoposide and ceramide treatment, whereas MEK inhibition increased the viability of ceramide treated cells. In A431 the MEK inhibitor modestly increased viability in control and taxol treated cells. These results are only in part consistent with the apoptosis assays presented in Figure 3, suggesting that the drugs have multiple modes of action. Therefore, we further tested other biological parameters.

All drugs have in common that cells feature high ERK and JNK activity around 18 h after treatment. As shown in Figure 4, taxol and ceramide cause cells to accumulate in the G₂/M phase of the cell cycle at this time point with the exception of ceramide treated HeLa cells, which retained a normal cell cycle profile. To investigate whether the MAPKs play a role in G₂/M arrest the MEK inhibitor PD98059 and the p38 inhibitor SB203580 were used. These inhibitors failed to rescue drug induced cell cycle blockades in A431 (Figure 5A) or HeLa cells (Figure 5B).

View larger version (49K): [in this window] [in a new window]   Fig. 5. The effect of ERK and p38 inhibitors on drug induced cell cycle blocks. Cells were treated (c: control; t: taxol; e: etoposide; C6: ceramide) and cell cycle profiles were analysed as described in Materials and methods. (A) A431 cells. (B) HeLa cells. Data show a typical example of two experiments.

 
The perhaps most important parameter for the efficacy of a chemotherapeutic drug is its long-term effect on cancer cell viability. Therefore, we used clonogenic assays to test whether the inhibition of ERK or p38 during a pulse of drug treatment would have a bearing on the outcome in a long-term assay that challenges the capacity of a cell to survive and thrive after an insult rather than querying a specific molecular mechanism (Figure 6). Cells were treated with drugs and MEK and p38 inhibitors as detailed in the Materials and methods section. After that they were seeded into normal growth medium and the outgrowth of cell colonies was scored 10–14 days later. All drugs significantly reduced colony formation. Curiously, the MEK inhibitor slightly increased the number of colonies in taxol treated HeLa cells suggesting that MEK inhibition was protective. In contrast, in A431 and to a lesser degree in MCF7 cells both the MEK and p38 inhibitors decreased the colony yield in response to etoposide.

	Discussion

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MAPK pathways have been implicated in the regulation of apoptosis as well as in the response to chemotherapeutic drugs (10). However, most of these studies were confined to one cell type or one particular class of drugs. The purpose of this study was to re-examine these issues on a broader scale and to evaluate whether the modulation of MAPK pathways could be employed to enhance the efficacy of chemotherapeutic drugs. The original perception was that stress-activated MAPKs, such as JNK and p38 promote apoptosis, whereas mitogen-activated MAPKs, such as ERK are protective (26). These early studies were carried out in PC12 cells, a rat pheochromocytoma cell line that is widely used as a model system for neuronal differentiation (29). These results stimulated further research into whether MAPK pathways are involved in the cellular response to chemotherapeutic drugs.

In particular, the activation of the ERK pathway has been linked to cell proliferation and survival. ERK can have a dual effect on proliferation (30). It was shown to stimulate proliferation by inducing the expression of cyclinD and hence activation of cell cycle kinases (31,32). In addition, ERK may phosphorylate and inactivate the p27^KIP cell cycle inhibitor protein (33,34). However, very high intensity ERK signalling halts the cell cycle by inducing the expression of cell cycle inhibitor proteins such as p21^Cip/Waf and p27^KIP (30). ERK can interfere with apoptosis on several levels. It can prevent the activation of caspases (35–37) and induce the expression of anti-apoptotic factors such as Mcl-1 (38) and IAPs, a family of small proteins that can bind to and inhibit caspases (39). Some of these functions may be related to ERK's ability to activate the CREB and NFB transcription factors that function in major survival pathways (40,41). However, ERK also has been reported to stimulate apoptosis in T-cells by enhancing Fas ligand expression (42) and to signal apoptosis in response to ceramide by preventing the inactivation of the pro-apoptotic bcl-2 family member BAD (43). The stress-activated kinases JNK and p38 at large have been associated with increasing apoptosis in response to external stress signals. The underlying mechanisms are less clear. JNK was reported to stimulate the expression of Fas ligand (44,45) and to phosphorylate and inactivate Bcl-2 (46). In prostate carcinoma cells Bcl-2 is protective against JNK induction of apoptosis (47). Bcl-2 also has a role as cell cycle inhibitor, and it has been reported that it needs to be phosphorylated and inactivated by JNK during physiological cell cycle progression (46). This role of JNK may be important for its requirement for cell proliferation in some cell types (48). Interestingly, the genetic removal of SEK-1, an immediate upstream activator kinase of JNK, promotes apoptosis of hepatocytes (49) and T-cells (50), revealing a dual role of JNK in the regulation of cell survival. Curiously, the genetic ablation of MEKK-1, an upstream activator of SEK-1, increases the sensitivity of murine embryonic stem cells to taxol (51). However, in a different cell line, chicken bursal B-cells, ablation of MEKK-1 protects from taxol induced apoptosis (52) indicating a cell type specific role of JNK in response to microtubule disrupting agents. p38 has been isolated as target of chemical inhibitors that interfered with the expression of pro-inflammatory cytokines including pro-apoptotic factors such as TNF (53). It can prevent cell cycle entry by down regulating the expression of cyclinD (32). It also mediates a G₂/M checkpoint by inactivating the Cdc25B phosphatase, which is needed to initiate G₂/M phase progression (54).

Taxol has been reported to activate JNK and p38 in a number of cell types (23). However, its effects on ERK are disparate. It appears that taxol inhibits ERK in suspension cells (38,55), but stimulates it in adherent cells (56–58). This may be related to the association of a pool of ERK with microtubules and the preferential activation of this pool by external signals in macrophages for instance (59). In our cells taxol activated ERKs, although the extent was modest, except in HeLa cells, and maximum activation occurred at the late time points between 18 and 36 h. Taxol induces a pronounced G₂/M arrest. As ERK is normally active during this phase (60,61) the late ERK activation could be an indirect consequence of the cell cycle block. Consistent with this explanation the late activation of ERKs by ceramide was stronger in A431 cells, where ceramide induced a G₂/M block, than in HeLa where the cell cycle was not inhibited. Interestingly, Fan et al. (62) observed that the effects induced by microtubules binding drugs, such as Bcl-2 phosphorylation, ERK and JNK activation, normally occur at the G₂/M phase boundary in synchronized KB-3 cells. They concluded that these drugs compromise cell viability by unduly extending and enhancing these physiological phenomena. Our results support such an interpretation. Another study showed that taxol toxicity was mitigated by the induction of high ERK activity and suggested that MEK inhibitors may enhance taxol effects in such cells (57). This hypothesis is supported by our findings that MEK inhibition increases PARP cleavage induced by taxol and etoposide in HeLa and to a lesser extent also in A431 cells. Both agents strongly induce ERK activity in these cells. Curiously, MEK inhibitors did not have unequivocal effects on other parameters of cell viability and apoptosis we tested. In the WST assay, which measures the short-term metabolic integrity of mitochondria, MEK inhibition slightly improved the basal level and reduced taxol inflicted damage in A431 cells and had no effect in HeLa cells. In the long term, clonogenic assay MEK inhibition enhanced colony reduction by etoposide in A431, but antagonized the taxol toxicity in HeLa cells. In this assay p38 inhibition also increased the etoposide toxicity in A431 cells. At present there is no obvious explanation for these discrepancies, but they highlight the complexity of assessing the effects of MAPK pathway manipulation as adjuvant tool for chemotherapy.

The most striking aspect of our study is the almost complete lack of correlation between the activation of MAPK pathways and drug effects measured in different assays. The clonogenic assay is perhaps the most relevant, because it gauges the long-term consequences of a single application of drug. This most closely reflects a clinical treatment situation where tumour cells are exposed to drugs in pulsed intervals rather than continuously. However, the clonogenic assay does not reveal the cause for a reduction of clones, which could be due to apoptosis, cell cycle delay or arrest, senescence, lower metabolic capacity or a combination thereof. Therefore, we used a number of short-term assays to evaluate these parameters individually. Chemotherapeutic drugs yielded clear and reproducible effects in any of these assays suggesting that they interfere with cell viability and proliferation on multiple levels. The manipulation of MAPK pathways alone or in combination with these drugs gave either no or rather diverse results. For instance, they had no effect on drug imposed cell cycle blocks, and diverse and small results in the WST assay. Thus, our results suggest that the original view of ERK promoting cell survival and JNK and p38 furthering apoptosis is too simplistic, and in this form cannot be exploited as a general tool to enhance chemotherapy. Rather our results suggest that the potential benefits of MAPK inhibition are confined to a subset of tumour cells, and hence must be individually tailored. They also show that currently no single experimental assay suffices to define these criteria in vitro, and that a battery of assays must be employed.

	Notes

 
⁴ To whom correspondence should be addressed at: The Beatson Institute for Cancer Research, Cancer Research UK, Switchback Road, Garscube Estate, Glasgow G61 1BD, UK Email: wkolch{at}beatson.gla.ac.uk

	Acknowledgments

 
S.B. was supported by a grant from La Roche Diagnostics.

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Received January 9, 2002; revised July 29, 2002; accepted August 15, 2002.

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