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Decreased Sensitivity to 1-O-Octadecyl-2-O-methyl-glycerophosphocholine in MCF-7 Cells Adapted for Serum-free Growth Correlates with Constitutive Association of Raf-1 with Cellular Membranes¹

Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3

	ABSTRACT

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We have previously shown that inhibition of MCF-7 cell proliferation by 1-O-octadecyl-2-O-methyl-glycerophosphocholine (ET-18-OCH₃) is linked to a drug-induced decrease in membrane Raf-1 levels and the subsequent inhibition of mitogen-activated protein (MAP) kinase activation in response to growth factor stimulation. We now report that adaptation of MCF-7 cells for growth in a serum-free formulation results in decreased sensitivity to growth inhibition by ET-18-OCH₃. The decrease in ET-18-OCH₃ sensitivity occurred progressively during the adaptation process and correlated with the presence of increasing amounts of inactive Raf-1 that stably associated with MCF-7 cell membranes. ET-18-OCH₃ sensitivity could be restored by growing the adapted cells in serum-containing medium, which resulted in the loss of membrane-associated Raf-1. In human normal mammary epithelial cells, which are insensitive to ET-18-OCH₃, Raf-1 was also associated with membranes in quiescent cells. In both cell types, incubation with ET-18-OCH₃ had no effect on the membrane-Raf-1 levels, suggesting that ET-18-OCH₃-induced reduction of Raf-1 levels in growth factor-stimulated MCF-7 cells is due to inhibition of Raf translocation. The activation and termination of the MAP kinase pathway in response to growth factors in the adapted MCF-7 cells and HNME cells occurred without changes to membrane Raf-1 levels. Because membrane translocation is not required to activate Raf in these cells, inhibition of Raf translocation by ET-18-OCH₃ subsequent to cell stimulation has no effect on the activation of the membrane-bound Raf and, consequently, the activation of the MAP kinase pathway. The ability of the cells to activate the MAP kinase pathway in the presence of the drugs enables them to resist the growth-inhibitory effects of the drug, leading to the observed ET-18-OCH₃ insensitivity of the cells.

	INTRODUCTION

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Aberrant transduction of growth signals in cells is thought to play an important role in the pathogenesis of cancer and other proliferative diseases. One of the intracellular pathways that transduces signals to the nucleus and other intracellular molecules from oncogenes and growth and differentiation factors is the MAP³ kinase cascade (1) . The protein kinase Raf-1 occupies a strategic position in this signaling pathway because it connects activated membrane molecules to the cytosolic protein kinases (2) . The activation of Raf-1 involves a complex series of events that are still not fully resolved. It is generally accepted that Raf-1 is cytosolic in unstimulated cells, but in response to cell stimulation, it is translocated to the membrane as a result of its interaction with Ras and is subsequently activated by events that may involve conformational changes, phosphorylation, and/or binding of lipid factors (3, 4, 5, 6, 7, 8, 9, 10) . Activated Raf-1 phosphorylates and activates MEK, which, in turn, phosphorylates and activates MAP kinase (2) .

ET-18-OCH₃ is the prototype of a diverse group of ether lipids collectively known as antitumor ether lipids that display antitumor properties in vitro and in vivo (11, 12, 13, 14) . Two characteristics of these compounds have generated considerable interest: (a) they achieve growth inhibition without interaction with cellular DNA and are therefore not mutagenic (12) ; and (b) they appear to inhibit the growth of cancer cells at concentrations at which they do not affect normal cells (12, 13, 14) and are therefore selectively cytotoxic. The basis for this selectivity is not clear. In a previous study, we demonstrated that in the MCF-7 cell line (breast adenocarcinoma), ET-18-OCH₃ inhibits mitogenic signaling via the MAP kinase pathway by decreasing the levels of Raf-1 associated with the membrane. This truncates the sustained phosphorylation of MEK that is needed to maintain MAP kinase in its phosphorylated and active state and the propagation of the mitogen signal (15) . The ET-18-OCH₃-induced decrease in the levels of membrane-associated Raf after MCF-7 cell stimulation (15) could be due to inhibition of the translocation of Raf from the cytosol or stimulation of the dissociation of Raf from the membrane. Because Raf-1 is ubiquitously expressed in cells, the ability of some cells to escape the inhibitory effects of ET-18-OCH₃ could be due to the utilization of Raf-1-independent mitogenic signaling pathways or activation of Raf by a mechanism that bypasses the step inhibited by ET-18-OCH₃.

In this study, we report that adaptation of MCF-7 cells for growth in serum-free medium renders the cells insensitive to the inhibitory effects of ET-18-OCH₃. We also show that during the adaptation process, increasing levels of Raf-1 associate with the cell membranes, allowing growth factors to activate Raf in the absence of its translocation from the cytosol. Consequently, inhibition of Raf translocation by ET-18-OCH₃ does not affect the level of Raf activation and activation of the MAP kinase cascade, resulting in little effect on cell proliferation.

	MATERIALS AND METHODS

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Materials.
HNME cells, mammary epithelial cell basal medium, and MEGM were obtained from Clonetics (San Diego, CA). DMEM, DMEM/F12, bovine pituitary extract, trypsin/EDTA, and GMS-X (1 mg/ml insulin, 0.55 mg/ml transferrin, 0.67 µg/ml sodium selenite, and 0.2 mg/ml ethanolamine) were obtained from Life Technologies, Inc. (Burlington, Ontario, Canada). Insulin, PGF₂, hydrocortisone, transferrin, fibronectin, soybean trypsin inhibitor, and calmidazolium were from Sigma (St. Louis, MO). EGF was a product of Collaborative Biomedical Products (Bedford, MA). Cyclic AMP-dependent protein kinase inhibitor peptide and protein kinase C inhibitor peptide were obtained from Bachem (Torrance, CA). FBS was purchased from HyClone (Logan, UT). ET-18-OCH₃ was provided by Medmark (Grunwald in Munich, Germany). [³H] ET-18-OCH₃ was obtained from Amersham (Oakville, Ontario, Canada) and repurified before use. Polyclonal anti-phospho-specific MAP kinase and anti-phospho-specific MEK antibodies were obtained from New England Biolabs (Mississauga, Ontario, Canada). Polyclonal anti-Raf-1 (C-12), anti-extracellular signal-regulated protein kinase 1 (C-16), anti-extra cellular signal-regulated protein kinase 2 (C-14), and anti-MEK1 (12-B) antibodies were products of Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal c-Raf-1 antibodies were purchased from Transduction Laboratories (Lexington, KY). Chemiluminescence detection reagent was purchased from Boehringer Mannheim (Laval, Quebec, Canada). Falcon tissue culture ware was obtained from Baxter Canlab (Winnipeg, Manitoba, Canada).

Adaptation of MCF-7 Cells for Growth in Serum-free Medium.
Confluent MCF-7 cells in 10% FBS-supplemented DMEM were subcultured into 10% FBS-supplemented DMEM diluted 1:1 with ACGM, which consisted of DMEM/F12 (1:1), 15 mM HEPES, and 24 mM NaHCO₃ (pH 7.4) supplemented with 1 µg/ml hydrocortisone, 20 ng/ml EGF, 5 µg/ml insulin, 7.5 µg/ml fibronectin, 25 µg/ml transferrin, 52 µg/ml bovine pituitary extract, 1% (v/v) GMS-X, 50 µg/ml gentamicin, and 50 ng/ml amphotericin with (ACGM⁺) or without (ACGM^-) 100 ng/ml PGF₂. At each subsequent passage, the FBS content was reduced by half by the addition of an equal volume of the appropriate ACGM until, after growing to 80% confluence in medium containing less than 0.1% FBS, the cells were subcultured directly into the ACGM. Cells growing in media with 0.625% FBS were subcultured into flasks that had been preincubated with 10% FBS-supplemented DMEM for 24 h. The media was completely removed by suction before the addition of the cells. The media were changed every 3 days. MCF-7 cells adapted for growth in ACGM⁺ were designated MCF-7Ad⁺, whereas cells adapted in ACGM^- (media without PGF₂) were designated MCF-7Ad^-. Stocks of the cell lines were frozen down in 10% FBS medium with 5% DMSO at -80°C for 3 h before storage in liquid N₂.

Cell Culture.
MCF-7 cells were grown in 10% FBS-supplemented DMEM. HNME cells were cultivated in MEGM according to the protocols provided by Clonetics. The adapted cells were subcultured by detachment with trypsin (0.025% trypsin/0.01% EDTA) followed by the addition of an equal volume of soybean trypsin inhibitor [0.25% (w/v)] and HEPES-buffered saline solution or the serum-free medium. The cells were centrifuged at 1000 x g for 5 min, resuspended in the appropriate media, and distributed in appropriate tissue culture ware that had been preincubated with 10% FBS-supplemented medium for 24 h as described above.

Isolation of Clones of MCF-7 Cells.
MCF-7 cells were trypsinized, dispersed in medium to a density of 100 cells/ml, and seeded in 150-mm dishes at a density of 10 cells/dish and incubated in a 5% CO₂ humidified atmosphere. After colonies were observed, individual colonies were trypsinized and subcultured into 6-well plates. Each colony was then expanded in T75 flasks, and stocks were frozen in liquid N₂.

Effect of ET-18-OCH₃ on Cell Proliferation.
The effect of ET-18-OCH₃ on the proliferation of MCF-7 cells was analyzed as described previously (16 , 17) . For cells growing in serum-free medium, the cells were detached with trypsin and pelleted as described above, and equal numbers were distributed into 24-well plates preincubated for 24 h with 10% FBS-supplemented DMEM/F12. When the cells were in log phase, medium containing ET-18-OCH₃ (0–30 µM) was added in 10% FBS-supplemented medium for MCF-7 cells and in the appropriate serum-free growth medium supplemented with 3 mg/ml BSA for the adapted MCF-7 cells and HNME cells. The cell numbers in representative wells were determined at the time of addition of ET-18-OCH₃. Forty-eight h later, the cell numbers were counted with a Coulter ZM counter, and the increase in the cell number was expressed as a percentage of controls without the drug. Cell viability was assayed by the trypan blue dye assay.

Uptake and Metabolism of ET-18-OCH₃ by Proliferating Cells.
[³H]ET-18-OCH₃ (specific activity, 0.1 µCi/µg/ml) was prepared in the required medium and added to proliferating cells growing in 6-well plates for uptake studies or 100-mm dishes for assessment of metabolism of the drug. At the selected times, the medium was removed, and the cells were washed twice with medium containing BSA (0.5 mg/ml). The cells were trypsinized, and after detachment, a known volume of medium containing 10% FBS was added, and the cells were transferred to tubes. After dispersion with a 21-gauge needle, aliquots were taken for cell counting, and the remainder was pelleted. Pellets from cells obtained from the 6-well plates were dissolved in 1% SDS in 0.3 M NaOH, and radioactivity was determined by scintillation counting after the addition of scintillant to the samples. The extent of metabolism of ET-18-OCH₃ was assessed using procedures described previously (18) .

Measurement of MAP Kinase and MEK Activity.
Quiescent cells were obtained by incubating proliferating cultures of MCF-7 cells in DMEM, MCF-7Ad⁺ cells in DMEM/F12, or HNME cells in mammary epithelial cell basal medium supplemented with 0.5 mg/ml fatty acid-free BSA until the 24 h increase in cell number was 10% or less. The cells were incubated with or without ET-18-OCH₃ for the stipulated times, washed twice, and stimulated for various periods. Subsequently, the cells were washed with ice-cold PBS, scraped into ice-cold buffer A [20 mM Tris-HCl (pH 7.4), 2 mM EGTA, 100 mM ß-glycerophosphate, 1 mM Na₃VO₄, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.2 mM aminoethylbenzylsulfonyl fluoride, 0.1 mM phenylmethylsulfonyl fluoride, 0.2 mM benzamidine, and 1 mM DTT], and disrupted by ultrasonication. Unbroken cells were pelleted by centrifugation at 7,000 x g for 10 min. The supernatant was then centrifuged at 200,000 x g for 30 min to separate the cytosol from the membrane fraction. Cell cytosol was flash-frozen and stored at -70°C until required. MAP kinase activity was assayed as the phosphorylation of MBP in the presence of protein kinase inhibitors (cyclic AMP-dependent protein kinase inhibitor peptide, calmidazolium, and protein kinase C inhibitor peptide) as described previously (15) . The activation of MAP kinase and MEK was assessed by Western blot analysis with anti-phospho-MAP kinase or anti-phospho-MEK antibodies using the protocols supplied with the product. Bound antibody was visualized by chemiluminescence and quantitated by densitometry.

Assessment of Membrane-associated Raf-1.
Cells were harvested as described above for the determination of MAP kinase activity. The pellet obtained after the high-speed centrifugation was suspended in extraction buffer A containing 1% Triton X-100 and 0.5% NP40 and sonicated, and the lysate was centrifuged at 200,000 x g for 30 min to obtain a clarified membrane extract (15 , 19) . The extract was removed and stored at -70°C. The Raf-1 levels were assessed by Western blot analysis with specific anti-Raf-1 antibodies.

Densitometric Analysis.
Quantitation of immunoblots and autoradiographs was achieved by densitometric analysis with a Model PDI 325oe high-resolution color scanner (Protein + DNA Imageware Systems, Huntington Station, NY) using the ImageMaster scanning program (Pharmacia).

	RESULTS

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The rationale for growing MCF-7 cells continuously in serum-free medium was to allow comparative studies with HNME cells to investigate the basis of ET-18-OCH₃ selectivity. Preliminary experiments revealed that MCF-7 cells were unable to grow in MEGM, which supports the growth of HNME cells. We were also unable to obtain long-term growth of MCF-7 cells in a formulation previously reported to support their growth (20) . Based on the latter report (20) and other reports (21 , 22) , two growth media, ACGM⁺ and ACGM^-, which differed only in the inclusion of PGF₂, were developed, and the MCF-7 cells were adapted for growth in the media to improve the growth rate. Adhesion and spreading of the cells was improved by preincubating the tissue culture ware with 10% FBS-supplemented medium for 24 h (22) . Fig. 1 shows the morphology of MCF-7, MCF-7Ad⁺, and MCF-7Ad^- cells. The adapted cells were more spindly and elongated than the parental cells. After growth in 10% FBS-supplemented medium for six passages, the morphology of the adapted MCF-7 cells was indistinguishable from that of the parental cell line.

View larger version (109K): [in this window] [in a new window]   Fig. 1. Morphology of the proliferating MCF-7, MCF-7Ad-, and MCF-7Ad+ cells. A, MCF-7; B, MCF-7Ad-; C, MCF-7Ad+.

2

View larger version (29K): [in this window] [in a new window]   Fig. 2. Effect of ET-18-OCH3 on the proliferation of MCF-7 cells, serum-free adapted MCF-7 cells, and HNME cells. a, equal numbers of MCF-7, MCF-7Ad+, MCF-7Ad-, and HNME cells were subcultured into 24-well plates. MCF-7 cells were grown in 10% FBS-supplemented DMEM. MCF-7Ad+, MCF-7Ad-, and HNME cells were grown in ACGM+, ACGM-, and MEGM, respectively. When the cells reached the log growth phase, they were incubated with growth medium containing ET-18-OCH3 (0–30 µM). All of the serum-free media were supplemented with 3 mg/ml BSA. Cells in representative wells were counted at the time of the addition of the compound. Forty-eight h after the addition of the compound, the cells were detached with trypsin, and the cell numbers were determined with a Coulter counter. The increase in cell number over day 0 for each concentration was expressed as a percentage of that in control wells that did not receive the compound. Each 24-well plate had its own control with no ET-18-OCH3. The results shown are the means ± SDs of three to five different studies with quadruplicate wells from four different experiments. b, MCF-7Ad+ cells were grown in 10% serum-containing medium for six passages. The effect of various concentrations (0–10 µM) of ET-18-OCH3 on the proliferation of the cells was then determined on MCF-7Ad+ cells as described above. The results shown are the means ± SD from quadruplicate wells from four different studies.

To eliminate the possibility that the insensitivity was due to the selection of a resistant population of cells in the initial culture, MCF-7 cells derived from single cell clones were obtained, and five arbitrarily selected clones were tested for their sensitivity to ET-18-OCH₃. As shown in Fig. 3a , all of the clones were sensitive to ET-18-OCH₃. The clones were adapted for growth in ACGM⁺ and tested for their sensitivity to ET-18-OCH₃. Like the population of mixed cells, the adapted clones were insensitive to ET-18-OCH₃ (Fig. 3b) , and sensitivity was restored after growth in 10% FBS-supplemented medium for six passages (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 3. Effect of ET-18-OCH3 on the proliferation of MCF-7 cell clones. a, 5 clones were arbitrarily selected from a population of 18 clones, each of which was derived from a single cell. The effect of ET-18-OCH3 on the proliferation of the clones (Clones 2–6) was investigated using the procedures described in the Fig. 2 legend. The results shown are the means ± SDs from quadruplicate wells from four different studies. b, the five MCF-7 clones selected for our studies (Clones 2–6) were each adapted for serum-free growth in ACGM+ by the procedures described in "Materials and Methods." The effect of ET-18-OCH3 on proliferation was investigated with each adapted MCF-7 clone. The results shown are the means ± SDs from quadruplicate wells from four different studies.

View larger version (63K): [in this window] [in a new window]   Fig. 4. Raf-1 levels in membranes from control and ET-18-OCH3-treated cells. a, membrane fractions were prepared from quiescent MCF-7Ad+ cells treated with or without ET-18-OCH3 (20 µM) for 5 h. After solubilization, aliquots of the lysate (15 µg) were separated by SDS-PAGE and Western blotted with polyclonal anti-Raf-1 antibodies. Bound antibody was visualized by enhanced chemiluminescence. Membrane fractions prepared from quiescent MCF-7 cells were also analyzed for comparison. The results shown are from three different MCF-7Ad+ cell preparations. b, quiescent MCF-7Ad+ cells were incubated with or without ET-18-OCH3 (20 µM) for 5 h, and the cells were washed and stimulated with EGF for various times. Membrane fractions were prepared, and Western blot analysis was conducted using monoclonal anti-Raf-1 antibodies as described above. The results shown are from a single experiment that is representative of the results obtained from four different cell preparations. c, quiescent MCF-7Ad+ cells were incubated with or without ET-18-OCH3 as described above, followed by stimulation with ACGM+ for various times. Membrane fractions were prepared, and Western blot analysis was conducted using monoclonal anti-Raf-1 antibodies. The results shown are from a single experiment that is representative of the results obtained from four different cell preparations.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Raf-1 levels and MAP kinase phosphorylation levels in unstimulated and EGF-stimulated HNME cell membranes. a, quiescent HNME cells were preincubated with or without 20 µM ET-18-OCH3 for 1.5 h. The cells were stimulated with EGF for varying periods and washed, and the membrane and cytosolic fractions were separated by centrifugation followed by solubilization of the membrane fraction as described previously. Aliquots of the soluble membrane fraction (15 µg of protein) were subjected to Western blot analysis with monoclonal anti-Raf-1 antibodies, and the bound antibody was visualized by enhanced chemiluminescence. The results shown are from a single experiment that is representative of those obtained with two different cell preparations. b, the cytosolic fractions were subjected to Western blot analysis with anti-phospho-MAP kinase or MAP kinase antibodies. Cytosolic protein (30 µg) was used for studies with phospho-MAP kinase antibodies, and 15 µg of protein were used for experiments with anti-MAP kinase antibodies. The results shown are from a single experiment that is representative of the results from two different cell preparations.

View larger version (47K): [in this window] [in a new window]   Fig. 6. Phosphorylation of MEK and MAP kinase in EGF-stimulated MCF-7Ad+ cells. a, quiescent MCF-7Ad+ cells were incubated with ET-18-OCH3 (20 µM) for 5 h. The cells were washed and stimulated with EGF for various times. Cytosolic fractions were obtained by centrifugation, and aliquots (50 µg of protein) were separated on 10% SDS-PAGE and Western blotted with anti-phospho-MEK antibodies. The results shown are from a single experiment that is representative of the results obtained from four different cell preparations. b, aliquots of the cytosolic fraction (30 µg of protein) from the experiments described in a were separated on a 10% SDS-PAGE gel and subjected to Western blot analysis using anti-phospho-MAP kinase antibody. Similar experiments with 15 µg of protein were probed with an anti-MAP kinase antibody. The results shown are from a single experiment that is representative of the results obtained with four different cell preparations. c, quiescent MCF-7Ad+ cells were treated with or without ET-18-OCH3 as described above in a, followed by stimulation with ACGM+ and isolation of the cell cytosol. Western blot analysis was performed with anti-phospho-MEK and anti-MEK antibodies. d, cytosolic fractions from the ACGM+-stimulated cells were also subjected to Western blot analysis with anti-phospho-MAP kinase antibodies. The results shown are from a single experiment that is representative of the results obtained with three different cell preparations.

View larger version (25K): [in this window] [in a new window]   Fig. 7. MAP kinase activity in stimulated MF-7Ad+, MCF-7, and HNME cells. a, quiescent MCF-7Ad+ cells were preincubated with or without 20 µM ET-18-OCH3 for 5 h. The cells were then washed and stimulated for varying periods with EGF (10 ng/ml). Cytosolic fractions were prepared, and MAP kinase activity was measured as the phosphorylation of MBP in the presence of protein kinase inhibitors with 1 µg of cytosolic protein. The results shown are the means ± SDs of triplicate incubations from a single experiment that is representative of the results with five different cell preparations. b, quiescent MCF-7Ad+ cells preincubated with or without 20 µM ET-18-OCH3 for 5 h were washed and stimulated with ACGM+. Cytosolic fractions were prepared, and an in vitro MAP kinase assay was conducted as described above. The results shown are the means ± SDs of triplicate incubations from a single experiment that is representative of the results from three different cell preparations. c, quiescent MCF-7 cells were preincubated with or without 20 µM ET-18-OCH3 for 3 h. The cells were then washed and stimulated for varying periods with EGF (10 ng/ml). Cytosolic fractions were prepared, and an in vitro MAP kinase assay was conducted as described above. The results shown are the means ± SDs of triplicate assays from a single experiment that is representative of the results obtained from three different cell preparations. d, quiescent HNME cells were incubated with or without 20 µM ET-18-OCH3 for 1.5 h. The cells were stimulated with EGF for varying periods, and cytosolic fractions were obtained by differential centrifugation. MAP kinase activity was determined using the in vitro assay described above. The results shown are the means ± SDs of triplicate incubations from a single experiment that is representative of the results obtained with two different cell preparations.

View larger version (40K): [in this window] [in a new window]   Fig. 8. Changes in ET-18-OCH3 sensitivity and membrane Raf-1 levels of MCF-7 cells during adaptation for growth in serum-free medium. a, MCF-7 cells were adapted for growth in ACGM+ as described in "Materials and Methods." During the adaptation process, the effect of ET-18-OCH3 on the proliferation of MCF-7 cells growing in medium containing 10%, 2.5%, 1.25%, 0.625%, 0.3125%, and 0.156% FBS was determined. The protein content of the drug-containing medium was adjusted with the addition of BSA to give a protein concentration of 3 mg/ml. The effect of ET-18-OCH3 on cell growth was determined after 48 h of incubation. The values represent the means ± SDs of quadruplicate determinations from four different studies. b, during the adaptation for growth in ACGM+ described above, cells growing in medium containing 5%, 2.5%, 1.25%, 0.62%, 0.31%, and 0% FBS were made quiescent, and the membrane fractions were prepared. The Raf-1 content in the solubilized membranes was determined by Western blot analysis using monoclonal anti-Raf-1 antibody. The results shown are from a single experiment that is representative of four different cell preparations.

The Increased Levels of Membrane-associated Raf-1 in MCF-7Ad⁺ Cells Are Linked to the Presence of PGF₂ in the Medium.

2

View larger version (36K): [in this window] [in a new window]   Fig. 9. Changes in membrane Raf-1 levels and ET-18-OCH3 sensitivity in MCF-7Ad- cells grown continuously in ACGM+. a, MCF-7Ad- cells passaged once in ACGM- were subsequently grown and passaged in ACGM+ for 0, 3, 6, 12, and 16 passages. Membrane fractions were prepared from quiescent MCF-7 cells, from quiescent MCF-7Ad- cells after 0, 3, 6, and 12 passages in ACGM+, and from MCF-7Ad+ cells. The membranes were solubilized and subjected to Western blot analysis with monoclonal anti-Raf-1 antibodies, followed by visualization with enhanced chemiluminescence. The results shown are from a single experiment that is representative of those obtained with four different cell preparations. b, the ET-18-OCH3 sensitivity of MCF-7Ad- cells grown in ACGM+ for 0, 6, 12, and 16 passages was determined using the procedures described previously. The results shown are the means ± SDs from quadruplicate wells from three different experiments.

	DISCUSSION

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In this study, we have demonstrated that adaptation of MCF-7 cells for growth in serum-free medium results in decreased sensitivity to ET-18-OCH₃ that is restored by growing the adapted cells back in serum-containing medium. Because our previous studies suggested that ET-18-OCH₃-induced inhibition of Raf-1 activation was responsible for the antiproliferative effect of the alkyllysophospholipid, we investigated whether the reversible ET-18-OCH₃ sensitivity observed was linked to changes in the status of Raf in the cells. This led to the discovery that adaptation of MCF-7 cells for growth in serum-free medium resulted in the constitutive association of Raf-1 with cell membranes in quiescent cells. Similar results were observed in quiescent HNME cells. The membrane-associated Raf in the quiescent cells was inactive because MEK and MAP kinase, which are downstream of Raf, were inactive in MCF-7Ad⁺ cells. This contrasts with the results of studies in which Raf-1 targeted to the membrane with CAAX motif was active in the absence of growth factor stimulation (23 , 24) . Activation of membrane-associated Raf-1 required stimulation of the cells by growth factors and resulted in the phosphorylation of MEK and MAP kinase, although the membrane Raf-1 levels were unchanged. These results indicate that there is a dissociation between Raf activation and translocation in these cells. This view is further supported by the fact that in MCF-7Ad⁺ cells stimulated with ACGM⁺ for 15 min, membrane Raf-1 levels did not decrease, although the signals from the growth factors were terminated as assessed by the return of MEK and MAP kinase phosphorylation/activation to resting levels (compare Fig. 4c and Fig. 6, c and d ). Thus, Raf activation and inactivation in these cells do not involve reversible translocation, in contrast to the situation in the parental MCF-7 cells (15) . The observed changes in the mechanism of activation of Raf-1 in these cells probably contribute to their relative ET-18-OCH₃ insensitivity. We have previously reported that ET-18-OCH₃ reduced the level of membrane-associated Raf-1 in MCF-7 cells by either inhibiting Raf translocation from the cytosol or stimulating Raf dissociation from the membrane (15) . Our current studies suggest that the diminished Raf levels observed in stimulated MCF-7 cells are likely due to the inhibition of Ras-mediated Raf translocation, based on the fact that uptake of ET-18-OCH₃ by MCF-7Ad⁺ or HNME cells to levels similar to those observed in MCF-7 cells did not diminish the Raf-1 levels in the membranes. In addition, the direct correlation between increasing membrane Raf-1 levels and resistance to ET-18-OCH₃ observed during the adaptation of MCF-7 cells to MCF-7Ad⁺ cells and during the growth of MCF-7Ad^- cells in ACGM⁺ also supports the notion that the level of membrane Raf-1 contributes to the insensitivity of the cells to ET-18-OCH₃. The results above provide additional evidence for our previous postulate that the effect of choline-containing alkyllysophospholipids on Raf activation may be a significant factor in their mechanism of inhibition of cell proliferation (15) and identify Raf-1 as a key intracellular target of these compounds, but by no means the only target (11) .

The mechanism responsible for the accumulation and stable association of inactive Raf-1 with cell membranes is not known. Our studies suggest that this may be related to the growth of cells in serum-free medium because the phenomenon was observed in three cells growing in serum-free medium (MCF-7Ad⁺, MCF-7Ad^-, and HNME cells). However, the levels of Raf-1 that accumulate are clearly influenced by specific factors in the medium. PGF₂ promoted Raf-1 accumulation in the membranes, because propagation of MCF-7Ad^- cells in PGF₂-containing medium resulted in a progressive accumulation of Raf-1 in the cell membranes. PGF₂ activates diverse signaling pathways including the Ras/Raf/MAP kinase pathway (25, 26, 27, 28, 29, 30, 31) ; however, it did not activate MAP kinase in quiescent MCF-7 and MCF-7Ad⁺ cells, although it is mitogenic in these cells (data not shown). Because chronic PGF₂ exposure was required to induce the accumulation of Raf-1 in the membranes, the mechanism may have little to do with those observed in response to acute stimulation. It is worth pointing out that if the membrane localization of Raf-1 in HNME cells is a consequence of its culture in serum-free medium, then the ability to cause Raf accumulation is not restricted to PGF₂ because MEGM does not contain PGF₂. On the other hand, the fact that HNME cells are primary cells raises the intriguing possibility that membrane localization of Raf in quiescent cells could be the norm for some cells because, after all, our current ideas on Raf distribution and activation are derived primarily from cell lines.

In conclusion, the studies described above have revealed that adaptation of cells for growth in serum-free medium may lead to changes in the subcellular distribution of specific intracellular molecules, leading to unforeseen consequences on cellular phenotype. In this instance, it resulted in the constitutive and stable association of Raf-1 with cell membranes; consequently, Raf-1 underwent activation and inactivation without dissociating from the membrane, contrary to current paradigms on the regulation of Raf activity (3) . Our results also suggest that the membrane localization of Raf could contribute to the ability of some cells to escape the growth-inhibitory effects of ET-18-OCH₃.

View larger version (24K): [in this window] [in a new window]   Fig. 10. Uptake and incorporation of ET-18-OCH3 into proliferating mammary cells. Proliferating MCF-7, MCF-7Ad-, MCF-7Ad+, and HNME cells were incubated with [3H]ET-18-OCH3 (10 µg/ml; 0.1 µ Ci/µg) for various times. The cells were washed and detached with trypsin. After further washing and determination of the cell numbers, the cells were sedimented by centrifugation and dissolved in SDS/NaOH. The quantities of ET-18-OCH3 incorporated were determined by scintillation counting and calculation from the specific radioactivity. The values represent the means ± SDs of six different experiments.

	FOOTNOTES

¹ Supported by a grant from the National Cancer Institute of Canada with funds from the Canadian Cancer Society (to G. A.).

² To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Biology, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0W3. Phone: (204) 789-3758; Fax: (204) 789-3900;

³ The abbreviations used are: MAP, mitogen-activated protein; ACGM, adapted cell growth medium; ET-18-OCH₃, 1-O-octadecyl-2-O-methyl-glycerophosphocholine; EGF, epidermal growth factor; FBS, fetal bovine serum; MBP, myelin basic protein; MEK, MAP kinase/extracellular signal-regulated protein kinase; HNME, human normal mammary epithelial; MEGM, mammary epithelial cell growth medium; PGF₂, prostaglandin F2.

Received 12/15/98. Accepted 8/ 4/99.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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