Reactive oxygen species have recently been demonstrated to play a role in numerous cellular signal transduction pathways. Here we investigate the involvement of H2O2 in Raf-1-mediated differentiation in the human medullary thyroid carcinoma (MTC) cell line TT:ΔRaf-1:ER. Catalase, but not Cu/Zn superoxide dismutase,completely inhibited Raf-1-induced differentiation ofβ-estradiol-treated TT:ΔRaf-1:ER. In addition, catalase treatment down-regulated RET expression at both the mRNA and protein levels and induced apoptosis in the parental TT cell line and uninduced TT:ΔRaf-1:ER human MTC cells. These results implicate H2O2 as a downstream mediator of c-Raf-1-induced differentiation and as a survival factor in MTC cells.

Many cell processes are sensitive to ROS.3 ROS are generally considered cytotoxic, inducing both apoptosis and necrosis by oxidatively damaging cellular components. However, ROS at low concentrations have recently been demonstrated to play a role in numerous cellular signal transduction pathways (1, 2, 3). Stimulation of various mammalian cells with cytokines, phorbol esters,or growth factors, including platelet-derived growth factor and epidermal growth factor, can induce the generation of ROS that can then act as second messengers in signal transduction pathways (4, 5, 6, 7). The generation of superoxide anion has recently been shown to mediate mitogenic signaling in Ras-induced cell cycle progression and cellular transformation in NIH 3T3 fibroblasts (5). However, whether ROS play a role in Ras/Raf-mediated cell differentiation is not currently known.

In the present report, we have examined whether ROS can play a role in cell differentiation. As a model system for cell differentiation, we have used the TT cell line of human MTC. The TT cell line has been shown to undergo differentiation in response to activation of the Ras/Raf signal transduction pathway (8, 9, 10, 11). Thus,introduction of a v-Ha-ras oncogene into the TT cell line of human MTC was shown to induce a differentiation program including cessation of growth, alteration in cell morphology, and increased transcription of the calcitonin gene (8). In TT cells, this differentiation program can also be achieved by estradiol-induced activation of a stably introduced Raf-1:ER fusion construct, ΔRaf-1:ER (9, 10). This Raf-1-induced differentiation is accompanied by down-regulation of the RET proto-oncogene; however, down-regulation of RET is not required for differentiation to occur (11). In the present study, we examined whether ROS might play a role in modulating the growth or Raf-mediated differentiation of TT:ΔRaf-1:ER cells. We now show that catalase treatment, which blocks accumulation of hydrogen peroxide(H2O2), completely blocksΔRaf-1:ER activation-induced TT differentiation. In addition,catalase treatment down-regulates RET expression and induces apoptosis in both the parental TT cell line and uninduced TT:ΔRaf-1:ER human MTC cells. These results indicate that ROS may be necessary for survival, appropriate gene expression, and Ras/Raf-mediated cell differentiation in the MTC cell model and suggest that ROS may also be required for these functions in other cell types, both in vitro and in vivo.

Cell Culture.

Generation of the TT:ΔRaf-1:ER cell line from a human MTC and its maintenance have been described previously (10). TT:ΔRaf-1:ER cells were maintained in 0.25 mg/ml Geneticin (Life Technologies, Inc., Grand Island, NY)-supplemented phenol red-free RPMI 1640 (Life Technologies, Inc.) with 16% FCS (Sigma, St. Louis, MO). Activation was achieved by the addition of 1 μmβ-estradiol (Sigma) in ethanol to the media. Control cells were treated with an equal volume of ethanol alone.

Fluorescent Measurement of Intracellular Peroxides.

To assess the levels of intracellular ROS, flow cytometric analysis was performed using the oxidation-sensitive probe CM-H2DCFH-DA as described previously (12). TT cells treated with ethanol, β-estradiol, or both β-estradiol and catalase (Sigma) were harvested, washed three times with 1× PBS, and incubated with 10 μmCM-H2DCFH-DA (Molecular Probe) for 30 min at 37°C. After incubation, the cells were placed in ice, and the fluorescein level was analyzed by flow cytometry using a Becton Dickinson FACScan with excitation and emission settings of 495 and 525 nm, respectively.

Flow Cytometry.

Flow cytometric analysis of TT cells treated with ethanol,β-estradiol, or both β-estradiol and catalase was performed using the propidium iodide (Sigma) staining method as described previously (12). Stained nuclei were analyzed on a Becton Dickinson FACScan with an argon ion laser at an excitation wavelength of 488 nm.

Western Blot Analysis.

Total cell protein was prepared by trypsinizing cells. The cell pellet was lysed in 2× Laemmli buffer (0.125 m Tris, 20%glycerol, 4% SDS, 10% β-mercaptoethanol, and 0.006% bromphenol blue). The cell pellets were boiled for 5 min and electrophoresed through SDS-PAGE gels. Gels were transferred to Immobilon-P membrane(Millipore). Membranes were blocked overnight at 4°C in 10% BSA in TTBS (0.1 m Tris, 150 mm NaCl, and 0.1% Tween 20). Antibodies against the following proteins were purchased from the indicated suppliers: polyclonal antibody against PARP was obtained from Boehringer Mannheim (Indianapolis, IN); mAb against human bcl-2 was obtained from DAKO (Carpinteria, CA); and mAb against β-actin was obtained from Sigma. Western blot analysis of RET protein was performed as described previously (10), and Western blot analysis of phospho-ERK-1/2 and ERK-1/2 was performed as described in Ref. 11 using total protein from 1 ×106 cells with affinity-purified polyclonal antibody against the COOH-terminal of RET (Santa Cruz Biotechnology,Santa Cruz, CA), anti-phospho-ERK-1/2, and ERK-1/2 polyclonal antibody(New England Biolabs, Beverly, MA). TT cell lysates treated with ethanol, β-estradiol, or both βestradiol and catalase were resolved by SDS-PAGE and transferred to Immobilon-P membrane(Millipore). Immunoblot analysis was performed with horseradish peroxide-conjugated antimouse or antirabbit IgG using the enhanced chemiluminescence Western blotting detection reagents from Amersham(Buckinghamshire, United Kingdom).

RNase Protection Assay.

Riboprobes were generated for RET and human GAPDH as described previously (11). Ten μg of RNA were used, and approximately 1 × 106 cpm of RET probe and 1 × 104 cpm of GAPDH probe were added to each reaction.

The Production of H2O2 during Raf-1-mediated Differentiation of TT Cells.

To examine the involvement of ROS in Ras/Raf-mediated differentiation,we have used the human MTC cell line TT:ΔRaf-1:ER, in which an activatable ΔRaf-1:ER fusion protein is constitutively expressed, as a model system. In this construct, the fusion protein is inactive until exposed to β-estradiol. On treatment of the cells with 1μ m β-estradiol, the fusion protein is activated immediately, and the cells are differentiated within 24–48 h. As shown previously, this differentiation program was characterized by morphological changes including cell rounding (Fig. 1,A) and by biochemical changes including a 4-fold increase in transcription of the differentiation marker calcitonin (Fig. 1 B). Treatment of the parental TT cells with 1 μm β-estradiol does not induce differentiation.

We first examined whether endogenous H2O2 levels were altered during Raf-1-induced differentiation, using CM-H2DCFH-DA fluorescence as an indicator of endogenous H2O2 levels. Cells readily take up CM-H2DCFH-DA, and its acetate groups are cleaved by intracellular esterase. Subsequent oxidation produces a fluorescent adduct. The addition of 1μ m β-estradiol to TT:ΔRaf-1:ER cells for 24 h to activate the ΔRaf-1:ER fusion construct increased the CM-H2DCFH-DA fluorescence level significantly compared with the ethanol-treated control cell populations (Fig. 2), indicating increased H2O2 levels during Raf-1-induced differentiation of MTC cells.

Inhibition of Raf-1-induced Differentiation by Catalase Activity.

The demonstration that intracellular H2O2 concentration is increased during Raf-1-induced differentiation suggested that H2O2 may act as a second messenger in Raf-1-induced differentiation of TT:ΔRaf-1:ER cells. Therefore, we examined whether this increased generation of H2O2 is required for the differentiation of TT:ΔRaf-1:ER cells. To examine the effect of blocking ROS production, we treated TT:ΔRaf-1:ER cells with the H2O2-catalyzing enzyme catalase during β-estradiol-induced differentiation. Catalase treatment significantly reduced Raf-1-induced CM-H2DCFH fluorescence to levels similar to those seen in ethanol-treated control cells (Fig. 2). Cotreatment of cells with 500 units/ml catalase for 48 h completely inhibited Raf-1-induced differentiation of β-estradiol-treated TT:ΔRaf-1:ER cells. Cells remained flat and adherent to the flask, with a morphology identical to that of ethanol-treated control TT:ΔRaf-1:ER cells (Fig. 1,A). Also, the expression of mRNA for the differentiation marker calcitonin was similar to that of ethanol-treated control TT:ΔRaf-1:ER cells (Fig. 1,B). In contrast, cotreatment of cells with 500 units/ml heat-inactivated catalase did not inhibit Raf-1-induced differentiation of TT:ΔRaf-1:ER cells (Fig. 1 A). Treatment with Cu/Zn SOD for 48 h did not block Raf-1-induced differentiation of TT:ΔRaf-1:ER cells (data not shown). However, unlike peroxide, superoxide does not readily cross the membrane; thus, it is possible that no effect of Cu/Zn SOD could be observed. Taken together, these findings support that H2O2 and/or other ROS are necessary components in the Raf-1-induced differentiation of TT:ΔRaf-1:ER cells. To determine whether H2O2 alone could induce a differentiation in TT:ΔRaf-1:ΔER cells, TT:ΔRaf-1:ER cells were exposed to various concentrations of H2O2 (10–1000μ m). Treatment of TT:ΔRaf-1:ER cells with H2O2 alone did not induce differentiation; however, at concentrations of >500μ m, treatment with H2O2 induced cell death (data not shown). Thus, intracellular H2O2 seems to be necessary,but not sufficient, for Raf-1-induced differentiation, suggesting that additional signaling components are required for TT:ΔRaf-1:ER differentiation.

Raf-1-induced ERK Activation and Cell Growth Arrest.

Raf mediates its effects in part by activation of a cascade of kinases including MEK-1/2 and ERK-1/2. In the TT:ΔRaf-1:ER cells, when the Raf-1 fusion kinase is activated by treatment with 1 μmβ-estradiol for 48 h, phosphorylation of MEK-1/2 and ERK-1/2 occurs (Ref. 13; Fig. 3). This phosphorylation cascade is required for differentiation (11). Although the addition of catalase in TT:ΔRaf-1:ER cells blocked β-estradiol-mediated morphological differentiation and the differentiation marker calcitonin (Fig. 1,B), it did not affect the phosphorylation of ERK-1/2 (Fig. 3). These data suggest that the role of H2O2 is either downstream to ERK-1/2 or in a pathway parallel to ERK-1/2. As reported previously, Raf-1-induced differentiation in TT:ΔRaf-1:ER cells is accompanied by terminal growth arrest (11). This arrest was cell cycle specific in G1 (Fig. 4 A; control cells,G1 = 80.7%, S phase = 13.1%, and G2-M = 6.2%; β-estradiol-treated cells,G1 = 95.9%, S = 1.9%, and G2-M = 2.2%). Blocking differentiation of TT:ΔRaf-1:ER cells by treatment with catalase did not relieve the cell cycle arrest.

Apoptosis Induced by Catalase in the Absence of Raf-1 Induction.

Treatment of the parental TT (data not shown) and uninduced TT:ΔRaf-1:ER cells with catalase resulted in accumulation of cells in G1, along with appearance of a sub-G1 peak, suggesting the induction of apoptosis(Fig. 4,A). The induction of apoptosis in the parental TT cells (data not shown) and uninduced TT:ΔRaf-1:ER cells by catalase was confirmed by observation of apoptotic morphological changes including a loss of adherence to the tissue culture flask, cell rounding, membrane blebbing, and nuclear fragmentation (data not shown). Also, catalase-induced apoptosis was detected by the cleavage of PARP into a Mr 85,000 fragmentation product at 48 h (Fig. 4 B). In contrast,treatment with 500 units/ml heat-inactivated catalase or with Cu/Zn SOD did not induce cell death in TT:ΔRaf-1:ER cells, indicating that it is the activity of catalase that induced cell death.

Down-Regulation of RET by Catalase in the Absence of Raf-1 Induction.

Activating mutations in the RET tyrosine kinase gene have been described in both sporadic and inherited cases of MTC (14, 15, 16). The TT cells harbor a Trp634 activating mutation in the RET gene (17). As reported previously (13), the addition of 1 μm β-estradiol to TT:ΔRaf-1:ER cells for 48 h inhibited RET expression at both the mRNA and protein levels, whereas ethanol-treated control TT:Raf-1:ER cells expressed the RET gene at both the mRNA and protein levels (Fig. 5,A). In the present study,catalase treatment for 48 h also inhibited RET expression at both the mRNA and protein levels in control (uninduced) TT:ΔRaf-1:ER cells(Fig. 5, A and B). Treatment of control TT:ΔRaf-1:ER cells with Cu/Zn SOD for 48 h did not inhibit RET expression at either the mRNA or protein level (data not shown). These data suggest that intracellular H2O2 and/or other ROS may play a pivotal role in mediating RET expression.

Stimulation of various mammalian cells with a wide variety of biological response modifiers, including cytokines, phorbol esters, or growth factors, can induce the generation of ROS, which can then act as second messengers in signal transduction pathways. Here we demonstrate that the intracellular H2O2concentration is increased during Raf-1-induced differentiation of TT:ΔRaf-1:ER cells and that blocking this increase by catalase treatment completely inhibits Raf-1-induced differentiation. Catalase treatment also down-regulates RET oncogene expression at both the mRNA and protein levels and induces programmed cell death in uninduced TT:ΔRaf-1:ER cells. Thus, these results imply that ROS are necessary for cell survival, cell proliferation, and Raf-1-induced differentiation in MTC cells. These results further suggest that ROS may be necessary for these cellular functions in other systems as well.

It should be noted that exogenous catalase is thought to act on endogenously produced H2O2in at least two ways. First,H2O2 is a long-lived,readily diffused ROS, and, once outside the cell, it can be detoxified by the exogenous enzyme (18). Alternatively, a cell type-specific accumulation of exogenous catalase through a receptor-mediated, energy-dependent system has been reported previously (19). It is not currently known which mechanism(s) is operative in the MTC cells. However, the results using CM-H2DCFH demonstrating that the addition of catalase reduces the amount of fluorescence in β-estradiol-treated cells to near control levels are completely consistent with the hypothesis that exogenous catalase treatment leads to a decrease in endogenous H2O2 (Fig. 2). The source of the endogenously produced H2O2 in the MTC cells is currently under investigation.

The mechanism by which ROS function downstream of Raf-1 in a signal transduction pathway leading to MTC cell differentiation remains unknown. ROS have been implicated in modulation of the Raf signal transduction pathway. In rat vascular smooth muscle cells,platelet-derived growth factor signaling results in rapid phosphorylation of mitogen-activated protein kinase via the Raf/MEK phosphorylation cascade, and this phosphorylation is inhibited by catalase treatment. However, in our system, catalase treatment did not affect the Raf-1-induced mitogen-activated protein kinase activity(Fig. 3). The activity of several transcription factors, which may be downstream effectors of the Raf signal transduction pathway, appears to be modulated by ROS, although the mechanism of their control is unclear. Thus, H2O2 has been implicated in activation of the transcription factor nuclear factor κB in mammalian cells, probably by increased phosphorylation of IκB (20), and activation of several transcription factors, including Elk-1, p53, and activator protein 1, has been reported to be modulated by redox regulation (2).

The catalase-mediated down-regulation of RET gene expression may be important for MTC biology. Mutational activation of the RET tyrosine kinase has been suggested to stimulate the initial growth leading to tumor development in many cases of MTC. Silencing of RET occurred during both Raf-1-mediated differentiation and catalase-induced apoptosis. Although it has recently been shown that continued expression of RET was insufficient to block Raf-1-induced differentiation of MTC (11), RET expression may be required for continued cell growth in MTC cells. If so, the mechanisms by which catalase leads to down-regulation of RET may be important targets for control of MTC in patients.

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 in part by NIH, NCI Grants CA57545, CA51085, and CA47480.

                
3

The abbreviations used are: ROS, reactive oxygen species; SOD, superoxide dismutase; CM-H2DCFH-DA,5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate;GAPDH, glyceraldehyede 3-phosphate dehydrogenase; MEK,mitogen-activated protein kinase kinase; MTC, medullary thyroid carcinoma; PARP, poly(ADP)-ribose polymerase; ER, estrogen receptor;ERK, extracellular signal-regulated kinase; mAb, monoclonal antibody.

Fig. 1.

The effects of catalase on Raf-1-induced differentiation in the TT:ΔRaf-1:ER cell line of human MTC. A, the effects of catalase on Raf-1-induced morphological changes of TT:ΔRaf-1:ER cells. TT:ΔRaf-1:ER cells were treated with 1 μm β-estradiol with or without 500 units/ml catalase for 48 h. Ethanol-treated control cells remain flat and adherent. Both ethanol- and catalase-treated cells remain flat, but catalase-treated cells lose viability and become detached from the flask. β-Estradiol-treated cells are rounded. Cells remain flat and adherent when treated with either β-estradiol and catalase or ethanol and heat-inactivated catalase but were rounded when treated with β-estradiol and heat-inactivated catalase. The results illustrated are typical of eight independent experiments. B, the effects of catalase on the Raf-1-induced differentiation marker calcitonin of TT:ΔRaf-1:ER cells. TT:ΔRaf-1:ER cells were treated with ethanol, ethanol and catalase,β-estradiol, or β-estradiol and catalase for 48 h. Total RNA was extracted and analyzed by blotting with cDNA probes specific for calcitonin. The same blot was then probed with a human GAPDH cDNA as a loading control, and the ratios of the amount of differentiation marker mRNA to that of differentiation marker GAPDH were calculated. The illustrated Northern blot results are representative of two independent trials with nearly identical results. In the bar graph,each bar represents the mean of the two experiments, and the range of the two experiments is indicated.

Fig. 1.

The effects of catalase on Raf-1-induced differentiation in the TT:ΔRaf-1:ER cell line of human MTC. A, the effects of catalase on Raf-1-induced morphological changes of TT:ΔRaf-1:ER cells. TT:ΔRaf-1:ER cells were treated with 1 μm β-estradiol with or without 500 units/ml catalase for 48 h. Ethanol-treated control cells remain flat and adherent. Both ethanol- and catalase-treated cells remain flat, but catalase-treated cells lose viability and become detached from the flask. β-Estradiol-treated cells are rounded. Cells remain flat and adherent when treated with either β-estradiol and catalase or ethanol and heat-inactivated catalase but were rounded when treated with β-estradiol and heat-inactivated catalase. The results illustrated are typical of eight independent experiments. B, the effects of catalase on the Raf-1-induced differentiation marker calcitonin of TT:ΔRaf-1:ER cells. TT:ΔRaf-1:ER cells were treated with ethanol, ethanol and catalase,β-estradiol, or β-estradiol and catalase for 48 h. Total RNA was extracted and analyzed by blotting with cDNA probes specific for calcitonin. The same blot was then probed with a human GAPDH cDNA as a loading control, and the ratios of the amount of differentiation marker mRNA to that of differentiation marker GAPDH were calculated. The illustrated Northern blot results are representative of two independent trials with nearly identical results. In the bar graph,each bar represents the mean of the two experiments, and the range of the two experiments is indicated.

Close modal
Fig. 2.

The production of H2O2during Raf-1-mediated differentiation of TT:ΔRaf-1:ER cells. H2O2 levels were determined by analysis of CM-H2DCFH fluorescence in control cells treated with ethanol, in cells treated with 1 μm β-estradiol, and in cells treated with 1 μm β-estradiol and 500 units/ml catalase for 24 h. The X axis represents log F1 fluorescence intensity; the Y axis represents cell number. In each experiment, 10,000 cells were analyzed.

Fig. 2.

The production of H2O2during Raf-1-mediated differentiation of TT:ΔRaf-1:ER cells. H2O2 levels were determined by analysis of CM-H2DCFH fluorescence in control cells treated with ethanol, in cells treated with 1 μm β-estradiol, and in cells treated with 1 μm β-estradiol and 500 units/ml catalase for 24 h. The X axis represents log F1 fluorescence intensity; the Y axis represents cell number. In each experiment, 10,000 cells were analyzed.

Close modal
Fig. 3.

The effects of catalase on Raf-1-induced ERK activation in TT:ΔRaf-1:ER cells. TT cells were harvested after treatment with β-estradiol with or without catalase for 48 h, and cell lysates were prepared and analyzed by Western blot using anti-phospho-ERK mAb.

Fig. 3.

The effects of catalase on Raf-1-induced ERK activation in TT:ΔRaf-1:ER cells. TT cells were harvested after treatment with β-estradiol with or without catalase for 48 h, and cell lysates were prepared and analyzed by Western blot using anti-phospho-ERK mAb.

Close modal
Fig. 4.

The effects of catalase on cell cycle distribution and apoptosis in Raf-1-induced differentiation of TT:ΔRaf-1:ER cells. A, flow cytometric analysis of cell cycle distribution. Cells were harvested after treatment withβ-estradiol with or without catalase treatment for cell cycle distribution analysis using propidium iodide. Histograms show the relative DNA content on the X axis and the number of cells on the Y axis. In each experiment, 10,000 cells were analyzed. B, catalase-induced PARP cleavage in TT:ΔRaf-1:ER cells. Cleavage of PARP to a Mr 85,000 fragment was induced by the addition of 500 units/ml catalase to TT cells of a human MTC for 48 h. Cell lysates were prepared and analyzed by Western blot using anti-PARP mAb.

Fig. 4.

The effects of catalase on cell cycle distribution and apoptosis in Raf-1-induced differentiation of TT:ΔRaf-1:ER cells. A, flow cytometric analysis of cell cycle distribution. Cells were harvested after treatment withβ-estradiol with or without catalase treatment for cell cycle distribution analysis using propidium iodide. Histograms show the relative DNA content on the X axis and the number of cells on the Y axis. In each experiment, 10,000 cells were analyzed. B, catalase-induced PARP cleavage in TT:ΔRaf-1:ER cells. Cleavage of PARP to a Mr 85,000 fragment was induced by the addition of 500 units/ml catalase to TT cells of a human MTC for 48 h. Cell lysates were prepared and analyzed by Western blot using anti-PARP mAb.

Close modal
Fig. 5.

The effects of catalase on RET expression. Cell lysates and total RNA were prepared from TT:ΔRaf-1:ER cells treated with 1 μm β-estradiol with or without 500 units/ml catalase for 48 h. Both catalase and activation of Raf-1 induced down-regulation of RET expression at both the mRNA and protein levels. A, RNase protection analysis of RET message in TT:ΔRaf-1:ER cells. B, Western blot analysis of RET in TT:ΔRaf-1:ER cells.

Fig. 5.

The effects of catalase on RET expression. Cell lysates and total RNA were prepared from TT:ΔRaf-1:ER cells treated with 1 μm β-estradiol with or without 500 units/ml catalase for 48 h. Both catalase and activation of Raf-1 induced down-regulation of RET expression at both the mRNA and protein levels. A, RNase protection analysis of RET message in TT:ΔRaf-1:ER cells. B, Western blot analysis of RET in TT:ΔRaf-1:ER cells.

Close modal

We thank Dr. Bertrand Tombal for expert assistance with morphological studies and Jim Flook for expert technical assistance with fluorescence-activated cell-sorting analysis.

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