Dietary energy restriction inhibits ERK but not JNK or p38 activity in the epidermis of SENCAR mice
Yinghui Liu1,
Ellen Duysen2,
Ann L. Yaktine2,3,
Angela Au1,
Weiqun Wang1 and
Diane F. Birt1,2,4
1 Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011 and
2 Eppley Institute for Research in Cancer and Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA
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Abstract
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Ongoing studies in our laboratory have demonstrated that dietary energy restriction (DER) inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 transcription factor binding to DNA in the epidermis of SENCAR mice. To dissect the specific signal transduction pathways through which DER inhibits the AP-1:DNA binding, we analyzed the activities of three major MAP kinases that lead to the induction of AP-1. The changes in ERK1 and ERK2 protein expression and phosphorylation were further characterized by western blot analysis. Female SENCAR mice were pre-fed ad libitum (AL) or 40% DER diet for 810 weeks. The kinase activities in mouse epidermis were determined by immune complex kinase assays at 0.5, 1, 4, or 6 h following treatment with 3.2 nmol TPA to the shaved dorsal backs. ERK activity at 1 h post-TPA treatment was nearly 5-fold (P < 0.005) above basal levels in AL mice while the increase was abolished in DER mice. The TPA-induced ERK activity in AL mice was accompanied by increased phosphorylation of ERK1 and ERK2 (P < 0.05), which was abrogated in DER mice. In addition, DER mice exhibited reduced expression of total ERK1 and ERK2 and higher proportions of ERK1 and ERK2 phosphorylation in comparison with AL mice (P < 0.05). JNK activity was decreased at 1 and 6 h but increased at 4 h (P < 0.05) post-TPA treatment. TPA did not change p38 kinase activity at the time points tested. Neither JNK nor p38 activity was altered by DER. Taken together, our results indicated for the first time that DER blocked the TPA stimulation of ERK activity and suggested that the inhibition of TPA-induced AP-1 activity by DER is likely through inhibition of ERK but not JNK or p38 kinase pathway.
Abbreviations: AL, ad libitum; DER, dietary energy restriction; DMBA, 7,12-dimethylbenz[a]anthracene; EGF, epidermal growth factor; ERK, extracellular signal-regulated protein kinase; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MBP, myelin basic protein; PKC, protein kinase C; TPA, 12O-tetradecanoylphorbol-13-acetate; UV, ultraviolet.
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Introduction
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Several lines of evidence, from both human and animal studies, suggest that excess energy intake increases the risk of cancer (1). Dietary energy (caloric) restriction (DER) is a well-documented inhibitor of both spontaneous and chemical-induced carcinogenesis in several species and with a variety of tumor types in animal studies (2). The effect is observed even when the energy-restricted animals were given a higher proportion of calories from fat than the controls (3,4). However, the mechanism(s) by which DER exerts its effects has not been fully elucidated. Studies have shown that DER may have a specific effect on various growth factors, oncogenes and tumor suppresser genes that are involved in the carcinogenic process (58).
Research in our laboratory is focused on the molecular mechanisms by which DER prevents skin carcinogenesis. We found that DER inhibits tumor promotion in the mouse skin carcinogenesis initiated by 7,12-dimethylbenz(a)anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) (3,9). We further observed attenuation of TPA-induced c-jun mRNA, c-Jun protein and AP-1:DNA binding in the epidermis of mice fed 40% less energy from fat and carbohydrate (2). Activation of AP-1 has been shown to play a key role in tumor promotion (1012). Transgenic mice carrying mutant c-jun with the transactivation domain deleted are resistant to DMBATPA induced skin papillomagenesis (13), demonstrating that AP-1 transactivation is required for skin tumor promotion in vivo. We therefore proposed that blockage of AP-1 transactivation is important in the observed inhibition effects of DER on the skin carcinogenesis.
Three distinct MAP kinase pathways have been identified in mammalian cells that converge to activate AP-1, namely, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAP kinase (14). ERK, activated by dual phosphorylation on both threonine and tyrosine residues, is preferentially stimulated by different growth factors and mitogens that lead to the induction of numerous downstream genes, such as transcription factors and other proteins regulating cell proliferation and differentiation (14,15). Although MAP kinase signaling pathways have been extensively investigated in cultured cell lines in the past decade, much less is known about the specificity and extent to which MAP kinases are activated in response to tumor promotion by TPA in vivo. We investigated the activities of ERK, JNK and p38 kinase in response to TPA treatment in the epidermis of SENCAR mice. Moreover, the impact of DER on the kinase activities was evaluated to establish the specific signaling pathways that may contribute to the inhibition of AP-1 activation and tumor promotion in the skin of DER mice.
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Materials and methods
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Animals and diets
Female SENCAR mice were obtained at 6 weeks of age from the National Institute of Health facilities at Frederick, MD. Animals were housed individually in a temperature and humidity-controlled room and kept on a 12 h light/dark cycle. The mice were randomly assigned to two dietary treatment groups: (i) AL, which were allowed free access to modified AIN-93 diet, or (ii) DER, which received a daily aliquot of a specific AIN formulated diet that restricted energy intake to 60% of that consumed by AL mice. The mice were given experimental diets 1 week after acclimation and maintained on their diets for 810 weeks. The composition of both diets is summarized in Table I
. DER diet was formulated by removing energy from carbohydrate and fat and was enriched in other nutrients such that DER mice would consume the same amounts of protein, vitamins, minerals and fiber as did their AL counterparts. Food intakes and body weights were recorded weekly. Feed was provided to AL mice weekly and to DER mice in daily aliquots that were based on the average daily food intake of AL mice for the previous week. All mice were freely provided tap water.
ERK immune complex kinase assay
Mice were treated with 3.2 nmol TPA in 200 µl acetone or with 200 µl acetone alone on the dorsal skin that was shaved 2 days before TPA treatment. Mice were killed by cervical dislocation 1 h after TPA treatment and the skin was removed and immediately immersed in liquid nitrogen. The 1 h time point was chosen because it was the time when maximal ERK induction by TPA was observed in the mouse epidermis in the preliminary study. The frozen skin was scraped with a single-edge razor blade and the epidermal cell scrapings were lysed in 1 ml cold lysis buffer containing 40 mM TrisHCl, 120 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.5% Triton-X 100, 10 mM sodium fluoride, 1 mM PMSF, 10 mM sodium pyrophosphate, 10 µg/ml leupeptin and 5 µg/ml aprotinin (pH 7.4). Homogenates were centrifuged at 14 000 g for 15 min at 4°C, and supernatants were then collected and total protein concentration was determined by standard BCA protein assay (BioRad, Hercules, CA). An equal amount of 500 µg total lysate protein was loaded and incubated for 4 h at 4°C with anti-ERK rabbit polyclonal antibody [sc-93 (recognizes both ERK1 and ERK2 protein); Santa Cruz Biotech, Santa Cruz, CA] that was pre-bound to protein A agarose beads (Gibco BRL, Grand Island, NY). The immunoprecipitates were collected by centrifugation followed by washing with lysis buffer then kinase buffer (40 mM TrisHCl pH 7.4, 20 mM MgCl2, 2 mM MnCl2, 25 µM ATP and 0.5 mM DTT). Kinase reaction was carried on the immunoprecipitates in the presence of 11 µg myelin basic protein (MBP; Gibco BRL), 0.7 mCi [
-32P]ATP (NEN Life Science, Boston, MA) and 15 µl kinase buffer. The reaction was incubated at 30°C for 30 min and the reaction products were subjected to a 15% SDSPAGE gel after completion. The dried gel was exposed to a phosphor screen and the radioactivity of the bands was visualized and quantified by a PhosphoImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuaNT software (Molecular Dynamics).
JNK and p38 kinase activity assay
Mouse epidermis was collected and homogenized as above using Triton lysis buffer containing 20 mM TrisHCl pH 7.4, with 137 mM NaCl, 25 mM B-glycerolphosphate, 2 mM EDTA, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 1% Triton X-100, 10% glycerol, 1 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 mM benzamidine and 0.5 mM DTT. Anti-JNK and anti-p38 kinase antibodies were purchased from Santa Cruz Biotech. JNK and p38 kinase activities were determined by immune complex kinase assays similar to ERK activity assay in the presence of kinase buffer (25 mM HEPES pH 7.4, with 25 mM ß-glycerolphosphate, 25 mM MgCl2, 0.1 mM sodium orthovanadate and 0.5 mM DTT), using either GSTc-jun (for JNK assay; Santa Cruz) or MBP (for p38 assay; Gibco BRL) as a substrate.
Western analysis of ERK1 and ERK2 proteins
An equal amount of 15 µg total epidermal lysate protein was electrophoresed on a 15% SDSPAGE gel and transferred onto PVDF membrane. ERK1 and ERK2 proteins were detected (antibody purchased from Santa Cruz Biotechnology, sc-93) by biotin/acidinalkaline phosphatase (AP) western blot detection system, following the manufacturer's instructions (Vector Laboratories, Burlingame, CA). Bands of ERK1 and ERK2 and their phosphorylated forms were separated by their distinct migration on the blot. NIH3T3 cells treated with 100 ng/ml TPA following 24 h of serum starvation were included as a positive control in the study. The blots were scanned and the density of each band was quantified by ImageQuaNT software (Molecular Dynamics). Protein levels were normalized as percentages of the average density of non-phosphorylated ERK2 (ERK2-NP) in the acetone/AL group. In addition, the proportions of ERK1 and ERK2 phosphorylation of each epidermis sample were calculated and expressed as the ratios of the band density of phosphorylated ERK1 or ERK2 (ERK1-P and ERK2-P) to that of the total ERK1/2 (ERK1/2-P plus ERK1/2-NP) in the same sample.
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Results
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Body weight
Body weights of ad libitum (AL) and energy restricted (DER) mice are shown in Figure 1
. Mice fed AL gained weight rapidly in the first 2 weeks and leveled off until the end of the study. The body weight of DER mice decreased steadily during the first 4 weeks after energy restriction and remained constant afterwards. At the end of the study, mice on the DER diet weighed significantly less than those of the AL group (23.2 ± 0.7 g versus 32.8 ± 0.7 g, P < 0.01). All of the animals survived to the end of the study.

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Fig. 1. Body weights of ad libitum and dietary energy restricted mice. Values represent the mean ± SEM of 16 mice/diet group. The SEM was within the symbol in instances where error bars are not shown. Mice were maintained on the experimental diets for 810 weeks. Statistical analysis at 8 weeks of experimental diet administration shows significant difference between AL () and DER ( ) mice (P < 0.01, single factor ANOVA).
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Dietary energy restriction inhibited TPA-induced ERK activity
The impact of DER on ERK activity was evaluated in epidermal lysates 1 h following TPA treatment of mouse skin. In the preliminary study, we tested ERK activation at 0, 1 and 4 h after 200 µl acetone or a tumor promoting dose of TPA (3.2 nmol in 200 µl acetone) treatment on the dorsal skin of AL mice. Maximal ERK activation in the epidermis was found 1 h after TPA treatment and was >4-fold above the acetone treated mice. This TPA-induced activation decreased to basal level by 4 h post-TPA treatment. In the subsequent study, we analyzed ERK activity in the epidermis 1 h after TPA or acetone treatment on AL or DER mice. The induction of ERK activity in Cos-1 cells by EGF was included in the study as a positive control (Figure 2A
, lanes 1 and 2). TPA dramatically increased ERK activity in the epidermis of AL mice (476 ± 227% over acetone controls, P < 0.005), while the induction was markedly abrogated in DER mice (Figure 2B
). DER did not change the basal level of ERK activity, since no difference was shown between AL and DER mice when treated with acetone alone.

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Fig. 2. Inhibition of TPA-induced ERK activity by dietary energy restriction. Mice were pretreated with experimental diets for 810 weeks before termination. ERK activity was measured by immune complex kinase assay at 1 h following TPA (dark bars) or acetone (Acet, dashed bars) treatment using MBP as a substrate. (A) A representative immune complex kinase assay showing ERK activity in the SENCAR mouse epidermis. Cos-1 cells with or without 10 nM EGF stimulation after 24 h of serum starvation were used as negative or positive controls in each experiment (lanes 1 and 2). The bands shown are radioactive phosphorylated MBP. (B) Quantitative analysis of ERK activation. Radioactivity of phosphorylated MBP as a measure of ERK activity was quantified using ImageQuaNT software (Molecular Dynamics). Values are expressed as percentages of the ERK activity over acetone/AL mice (mean ± SEM). DER significantly inhibited ERK activity induced by TPA (P < 0.001, n = 8 mice/group). *, Significant induction of ERK, P < 0.005.
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DER effect on the expression of ERK proteins in the mouse epidermis
We performed western blot analysis on the total lysate protein to assess the changes in ERK1 and ERK2 protein and their phosphorylation. Activation of ERK1 and ERK2 is characterized by a reduced mobility in SDSPAGE as a result of phosphorylation of specific threonine and tyrosine residues (16). A representative western blot (Figure 3A
) shows two distinctive bands representing non-phosphorylated ERK1 (ERK1-NP) and non-phosphorylated ERK2 (ERK2-NP) in an acetone/AL mouse sample (lane 4) and samples with two additional slower migrating bands representing phosphorylated ERK1 (ERK1-P) and phosphorylated ERK2 (ERK2-P) (lanes 25 and 7). Lanes 6 and 7 represent negative and positive controls, respectively, of NIH 3T3 cells treated with acetone or 100 ng/ml TPA following 24 h of serum starvation (Figure 3A
). Both ERK1-P and ERK2-P were elevated >2-fold in TPA/AL mice (227 ± 32 and 234 ± 28%, respectively, over acetone/AL mice, P < 0.005; Figure 3B
), suggesting that ERK1 and ERK2 phosphorylation both significantly contribute to ERK activation by TPA in the epidermis of SENCAR mice. ERK1-P and ERK2-P were not changed by TPA in DER mice (103 ± 13 and 119 ± 20%, respectively, over acetone/DER mice). In addition, DER mice displayed reduced levels of ERK1-NP and ERK2-NP (Figure 3B
), and thus reduced total ERK1 and total ERK2 (P < 0.05). Along with increased ERK activity, the ratios of ERK1 and ERK2 phosphorylation were upregulated by TPA in AL mice (P < 0.005; Table II
). Interestingly, DER mice displayed higher proportions of ERK1 and ERK2 phosphorylation compared with the basal ratio in acetone/AL mice (P < 0.05), and further the proportions were not changed by TPA treatment in DER mice (Table II
). It should be noted that since the total ERK1 and ERK2 protein levels were decreased in DER mice, the actual amount of ERK1 and ERK2 that were phosphorylated in these mice were not significantly different from those in acetone/AL mice (Figure 3B
). This observation was consistent with the result that DER did not change the basal ERK activity in the epidermis (Figure 2B
).

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Fig. 3. Effect of TPA and DER on the expression of ERK1 and ERK2 proteins in the epidermis of SENCAR mice. Equal amounts of whole epidermal cell lysate were separated by SDSPAGE and immunoblotted with anti-ERK antibody. The density of each of the bands was quantified by ImageQuaNT software (Molecular Dynamics). (A) A representative western blot showing expression of ERK1 and ERK2 proteins in the SENCAR mouse epidermis. NIH 3T3 cells with or without TPA stimulation after 24 h of starvation were shown as negative and positive controls of ERK1 and ERK2 phosphorylation and mobility shifts of phosphorylated ERKs (lanes 6 and 7). P, phosphorylated; NP, non-phosphorylated form. (B) Quantitative analysis of ERK1 and ERK2 expression. The protein levels are presented as the percentages of the average density of ERK2-NP protein in acetone/AL mice (mean ± SEM). Statistical comparisons were conducted by t-test within each protein. *, Significant reduction (P < 0.05) compared with acetone/AL mice. , Significant elevation (P < 0.05) compared with acetone/AL mice. n = 8 in each treatment group.
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JNK and p38 activity in the epidermis of SENCAR mice
In parallel studies, we tested whether JNK or p38 kinases, two other upstream activators of AP-1 transcription factor induction, were changed by TPA and DER. JNK and p38 MAP kinase activities in the epidermis were evaluated at different time points after TPA treatment in vivo. JNK activity was reduced by 23% (P < 0.05) and 26% (P < 0.05) after 1 and 6 h, respectively, of TPA treatment while a moderate induction was observed 4 h post-TPA treatment in comparison with the acetone treated mice (P < 0.05; Table III
). However, no significant difference of JNK activity was observed in AL mice compared with DER mice at each of the time points tested (Table III
). In a preliminary study, we measured p38 kinase activity at 0, 1 and 4 h of post-TPA treatment in the epidermis of AL mice. No significant induction was found at any of the times tested. P38 kinase activity analyzed 1h following TPA treatment in the current study was 110 ± 23, 92 ± 10 and 95 ± 6% in the TPA/AL, acetone/DER and TPA/DER group, respectively (as a percentage of the acetone/AL mice, mean ± SEM). P38 kinase activity was not significantly changed by either TPA or DER.
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Discussion
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Tumor promotion is an important step in multistage carcinogenesis and has been a major target of cancer prevention. Dietary energy restriction (DER), a well-documented carcinogenesis inhibitor in animal studies, has been shown to inhibit tumor promotion in the two-stage skin carcinogenesis model (3,9). In an attempt to understand the molecular mechanisms that are responsible for this inhibition of tumor promotion, we investigated the impact of DER on ERK, JNK and p38 kinase activities following a single tumor promoting dose of TPA in the epidermis of SENCAR mice. Our data demonstrated that TPA caused dramatic changes in ERK, minor changes in JNK and no alterations of p38 kinase activity in the epidermis of SENCAR mice in vivo. This observation is in agreement with a recent report that topical application of TPA on the ears of CF1 mice induced a rapid and sustained elevation of ERK activation but not JNK or p38 in vivo (17). Most importantly, we found that mice prefed with 40% DER diet for 810 weeks displayed significantly decreased TPA-induced ERK activity in the epidermis compared with AL controls. The reduction of ERK activity was accompanied by reduced levels of total ERK proteins in DER mice and a decrease in phosphorylation of ERK1 and ERK2 proteins in response to TPA. JNK and p38 MAP kinase activities, on the other hand, were not changed by DER diet. These data suggest that the observed inhibition of TPA-induced AP-1 activation by DER is through the inhibition of the ERK signaling pathway. Since JNK and p38 kinases were not altered in DER mice, these pathways do not appear to contribute significantly to the DER inhibition of AP-1 induction by TPA.
It is noteworthy that ERK displayed the greatest TPA induction (nearly 5-fold) among the three MAP kinases tested and dietary intervention was most apparent against the induced activity. Since there were only minor changes of JNK and no alterations of p38 activity after TPA treatment, these pathways may not have been as sensitive to the modulation effect of DER under the conditions we used in this study. Future studies will be required to determine if DER influences the induction of p38 using agents that stimulate this kinase. However, our intent was to determine the pathways that are modified by DER during TPA promotion and thus the focus of the present investigation was on TPA as a stimulator of AP-1 signaling.
Several lines of evidence have implicated the importance of ERK in cancer development. Certain tumor promotion responsive genes such as Ras (18), Raf-1 (19), PKC (20) and c-Jun (21) have been identified as major components in the ERK signaling in vitro (22). Constitutive activation of ERK has been observed in certain human tumors, including renal cancer (16), breast cancer (23,24) and prostate cancer (25). In those tumors, ERK is overexpressed and its activity is significantly higher in cancerous tissues as compared with normal surrounding tissues. On the contrary, expression of dominant negative ERK2 inhibited AP-1 transactivation and neoplastic transformation (26). Furthermore, a promotion-resistant mouse epidermal cell line displayed deficient ERK activity. Stable transfection of wild-type ERK2 into these cells restored their response to TPA and EGF for both AP-1 activation and cell transformation (27). These observations predict the importance of blocking ERK signaling as an effective way of preventing carcinogenesis. TPA has been known as a potent tumor promoter in mouse skin (28) and TPA induces ERK activity and cell proliferation in cultured cells (27,29). In our study, ERK1 and ERK2 activity was profoundly increased by TPA treatment in AL mice, while this elevation was abolished by DER. The results we reported here thus provide significant evidence that DER is a potent inhibitor of TPA-induced ERK activation in vivo, which may contribute significantly to the DER prevention of skin tumor promotion.
The reduced ERK activity in DER mice may be caused by inhibition of upstream regulatory signaling. We previously found that DER decreased the basal levels of PKC activity and expression of PKC
and PKC
isoforms in the epidermis of DER mice (30,31). Interestingly, although the levels of total ERK1 and total ERK2 protein in acetone/DER mouse skin were decreased in comparison with acetone/AL mouse, basal ERK activity was not decreased in DER mice. This apparent discrepancy may be explained by the compensating increased ratios of ERK1 and ERK2 phosphorylation in these mice. Apparently, other components in the signaling cascade or cross-talk from other pathways may be modulated to maintain the basal level of ERK activity and thus possibly maintain normal cell growth and differentiation in the epidermis of DER mice. Changes of Ras and Raf-1 expression in these mice merit further investigation. In addition, studies of the stability of ERK protein and its mRNA should be informative.
JNK and p38 pathways are primarily stimulated by the activation of cytokine receptors and cellular stress including UV irradiation (14). In mammalian cells, UV induces ERK, JNK and p38 MAP kinase pathways and their induction of AP-1 activation may play a critical role in UV-induced skin carcinogenesis (32,33). The importance of JNK modulation by TPA observed in this study is not known. Some studies suggest that ERK and JNK signaling pathways transduce opposing signals that control cell proliferation and apoptosis (3436). In mouse epidermal cells, introduction of the dominant negative mutant of JNK did not inhibit TPA-induced cell transformation, suggesting that JNK activation may not be required for TPA-induced cell transformation in this model (37). P38 MAP kinase has recently been implicated in the proliferative response in some cell lines (3840). P38 kinase activity was not changed by the TPA treatment in our study. Most importantly, neither JNK nor p38 was significantly changed by DER diet under the conditions used.
In line with our observation that TPA-induced AP-1 activity was reduced in energy restricted mice (2) and the requirement of AP-1 for mouse skin tumor promotion reported by another group (13), DER may exert its skin tumor preventive effect by inhibiting AP-1 activation through pathways dependent on ERK but independent of JNK or p38 kinase. In conclusion, our findings provide significant insights into the cellular mechanisms underlying the prevention of skin carcinogenesis by DER.
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Notes
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3 Present address: MGH Cancer Center, Molecular Oncology, Building 149, 13th Street, Charlestown, MA 02129, USA 
4 To whom correspondence should be addressed 
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Acknowledgments
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We thank Dr Janice E.Buss and her laboratory for assistance with the ERK phosphorylation analysis and Dr Jill C.Pelling for consultation with the JNK and p38 kinase assays. This work was supported by National Cancer Institute (RO1 CA77451) and American Institute for Cancer Research Grant (97B039). This paper is published as Journal Paper J-19219 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project no. IOW03360, and supported by Hatch Act and State of Iowa funds.
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Received October 13, 2000;
revised December 22, 2000;
accepted December 28, 2000.