Tumor promoters inhibit spontaneous differentiation of Friend

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 74, No. 7, pp , July 1977 Cell Biology Tumor promoters inhibit spontaneous differentiation of Friend erythroleukemia cells in culture (phorbol diesters/terminal differentiation/hemoglobin subunits/mouse erythroleukemia) GIOVANNI ROVERA, THOMAS G. O'BRIEN, AND LEILA DIAMOND The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pennsylvania Communicated by Hilary Koprowski, April 18,1977 ABSTRACT Clones of Friend erythroleukemia cells, characterized by the presence of 40-70% benzidine-positive cells synthesizing hemoglobin in the absence of inducing drugs, were treated with several phorbol diesters with a known range of tumor-promoting activity on mouse skin. Good correlation was found between the reported tumor-promoting activity of a particular phorbol diester and its ability to inhibit spontaneous erythroid differentiation in culture. The inhibition of differentiation by 12-O-tetradecanoyl-phorbol-13-acetate, the most active tumor promoter, was maximum after 4 days of treatment; this inhibition was reversed by removal of the phorbol diester no matter how long the period of treatment. Unlike control cells, which gradually revert to a po ulation with a low percentage of benzidine-positive cells, cels treated with 12-O-tetradecanoyl-phorbol-13-acetate retained a high potential for spontaneous differentiation. Friend erythroleukemia cells in culture consist of mixed populations of differentiating and undifferentiated cells (1). Undifferentiated cells, considered to be stem cells committed to erythroid differentiation, usually predominate (for reviews and discussion, see refs. 2 and 3). Clones with a high percentage of spontaneously differentiating cells can be isolated (4); these contain from 40 to 70% benzidine-positive (B+) cells at various stages of maturation. The uncommitted stem cell population in these clones tends to revert to a phenotype that gives rise to populations with lower percentages of differentiating cells. *t Despite the preferential reversion towards a more undifferentiated phenotype, these clones are excellent models for the study of phenotypic variation and spontaneous differentiation in culture. During a search for factors that might affect spontaneous differentiation, we tested diesters of the diterpene alcohol, phorbol, with a wide range of tumor-promoting activity in vivo (5-7). Tumor-promoting compounds are not themselves carcinogenic, but can induce skin tumors in mice that have been treated with a subcarcinogenic dose of a chemical carcinogen; their mechanism of action is not understood (for reviews, see refs. 6 and 8-10). We now report the positive correlation between the tumor-promoting activity of a given phorbol diester and its ability to inhibit spontaneous differentiation of Friend leukemia cells in culture. The observation supports the suggestion that tumor promoters act in normal cell differentiation (11, 12). ivo by interfering with MATERIALS AND METHODS Cell Cultures. Three spontaneously differentiating sibling clones, D7, D7/1, and D7/64, were obtained from suspension Abbreviations: B+, benzidine-positive; TPA, 12-0-tetradecanoylphorbol-13-acetate; PDD, phorbol-12,13-didecanoate; 4-a-PDD, 4- a-phorbol-12,13-didecanoate; PDB, phorbol-12,13-dibenzoate; PDA, phorbol-12,13-diacetate. * G. Rovera and G. Meier, unpublished data. t G. Rovera and C. A. Marsh, unpublished data. cultures of Friend virus-induced murine erythroleukemia cells (GM86 Clone 745) by cloning in methocel; these clones had 50-70% B+ cells when used in the experiments reported here (ref. 4; *). The cells were grown in suspension in 25-cm2 plastic flasks (Falcon or Costar) containing 10 ml of Eagle's minimal essential medium (Auto-Pow, Flow Labs) supplemented with 2 mm glutamine, 100 gg/ml of penicillin, 100 Ag/ml of streptomycin, and 15% fetal bovine serum (Flow Labs). (Two pretested lots of fetal bovine serum were used in all experiments.) The flasks were incubated horizontally in a humidified atmosphere with 10% CO2 in air. The cells were routinely inoculated at a concentration of 105 cells per ml and subcultured after 4-5 days when the cell concentration was 1.5 to 2 X 106 cells per ml. Evaluation of Differentiation. The percentage of differentiating cells was determined routinely by scoring B+ cells. The benzidine reaction was done on cell suspensions dispersed into multiwell trays (Falcon or Linbro) as described by Orkin et al. (13). Five to twenty minutes after addition of the benzidine reagent, 400 cells were scored by each of two observers. We determined the amount of hemoglobin in the cultures by washing approximately 5 X 106 cells with phosphate-buffered saline and lysing them with 1 ml of 0.3% Triton X-100 in 50 mm TrissHCl, ph 7.0/25 mm KCI/5 mm MgCI2/1 mm 2-mercaptoethanol (14). The lysate was centrifuged at 11,000 X g for min and the amount of hemoglobin was determined spectrophotometrically by absorbance at 414 nm, as described by Kabat et al. (14). The Triton/acidic urea gel electrophoresis system described by Zweidler and Cohen (15) for the separation of histones was used to separate the globin chains. Cells were labeled for 4 days with 0.5 ACi/ml of a mixture of 14C-labeled amino acids (New England Nuclear Corp.). Cytoplasmic lysates were prepared as described above and the amount of protein was determined by the procedure of Lowry et al. (16). To determine the specific activity, we precipitated 0.2-ml aliquots with 5% trichloroacetic acid. The precipitates were collected on GF/C glass filters, washed three times, and assayed for radioactivity using Omnifluor (New England Nuclear) as the scintillator. An aliquot of the sample (80,ug of protein in 50 gl) was denatured by mixing with an equal volume of a solution containing 4 M urea/5% acetic acid/5 mm 2-mercaptoethanol. After 0.02% pyronine Y was added, the sample was run for 6 hr at 140 V on a 12.5% acrylamide slab gel, 0.75 mm thick. Gels were stained with amido Black, destained electrophoretically, prepared for fluorography (17, 18), and exposed to x-ray film (Kodak RPO Mat) at -70 for 4 days. Chemicals. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) and 4-a-phorbol-12,13-didecanoate (4-a-PDD) were purchased from Consolidated Midland Corp., Brewster, NY. Phorbol- 12,13-dibenzoate (PDB), phorbol-12,13-diacetate (PDA), and phorbol-12,13-didecanoate (PDD) were a gift of R. K. Boutwell 2894

2 Cell Biology: Rovera et al. Proc. Natl. Acad. Sci. USA 74 (1977) j I A A 0 ~40 + M30) 0w X X 10-8 M 1.6 X 10-7 FIG. 1. Dose-response of clone D7/64 cells to phorbol and phorbol diesters. (0) Phorbol; (-) PDD; (0) 4--a-PDD; (3) PDA; (&) PDB; (U) TPA. Each point is the average of two to four cultures. of the McArdle Laboratory for Cancer Research, University of Wisconsin. The compounds were dissolved initially in acetone at a concentration of 1.6 X O-4 M and were stored in the dark at -0 until use; the final concentration of acetone in the medium was 0.1% or less. 70- A ~~B C 0~~~~~~~~~~~~~ 60*-0 RESULTS Response of Differentiating Friend Leukemia Cells to Phorbol Diesters. Cells from clone D7/64 were treated for 4 days with various concentrations of phorbol diesters and the B+ cells were scored (Fig. 1). The percentage of B+ cells in untreated control cultures was 72%. The smallest percentage of B+ cells occurred in cultures treated with 1.6 X 0-7 M TPA or PDD. TPA decreased the number of B+ cells even at a concentration as low as 1.6 X 10-9 M. PDB was not active at a concentration of 1.6 X 10-8 M, but at higher concentrations did decrease the percentage of B+ cells. PDA was slightly inhibitory, but only at the highest concentrations tested; 4-a-PDD, phorbol, and the solvent, acetone, had no effect on the percentage of B+ cells. At the concentrations tested, none of the phorbol diesters affected the doubling time of the cells when added to cultures containing 105 cells or more per ml; after 4 days the cell den Ua FI.22 feto0pobladpobo ises(. 0 A\ 10-M)o FIG. 2. Effect of phorbol and phorbol diesters (1.6 X 10O7 M) on the percentage of B+ cells in clones D7 (A), D7/1 (B), and D7/64 (C) as a function of time. (0) Control; (0) TPA; (ti) PDB; (o) PDA; (-) phorbol. %% FIG. 3. Effect of TPA on the percentage of B+ cells in clone D7. (0-0) Untreated cells; (O@---@0) cells treated with TPA (1.6 X 10O7 M). The arrows indicate when the cells were subcultured into medium with or without TPA. sities in control and treated cultures were the same (data not shown). The effect of several phorbol diesters (1.6 X 10-7 M) on three spontaneously differentiating clones over several days is shown in Fig. 2. All three clones showed a decrease in the percentage of B+ cells after treatment with TPA and PDB that was directly correlated with the length of time the cells were in contact with the compounds. Treatment with the same concentration of PDA was less effective in reducing the number of B+ cells, and treatment with phorbol for 4 days had no effect. Cultures of clone D7 were treated with TPA (1.6 X 10-7 M) at various times over a period of 25 days (Fig. 3). As would be expected because of the instability of the phenotype,* the percentage of B+ cells in the untreated control culture gradually declined from 65 to 30%. However, when these cells were treated with TPA at any time during the 25-day period, the percentage of B+ cells was reduced to a baseline of 5-10% within 4 days (Fig. 3). Long-Term Effects of TPA Treatment. Cultures of clone D7 with 62% B+ cells were exposed continuously to TPA (1.6 X 1O-7 M). After the initial decrease in the percentage of B+ cells, there was a gradual increase from a low of 5% after 5 days to a high of 17% after 18 days (Fig. 4). The cultures maintained a level of about % spontaneously differentiating cells during an additional 4-week period of TPA treatment (data not shown). Removal of the TPA from parallel cultures at different times during this period resulted in a rapid increase in the number a /D FIG. 4. Effects of continuous treatment with TPA and of removal of TPA on spontaneous differentiation of clone D7 cells. (o---o) Cultures without TPA; (*-*) cultures treated with TPA (1.6 X 10-7 M). The arrows indicate when the cultures were subcultured into medium with or without TPA. A

3 2896 Cell Biology: Rovera et al. ~ ~ ma10-2. \ A 7 - 'Zt _Y 10-3 '<l a FIG. 5. Effect of TPA on retention of acid-insoluble radioactivity by cells labeled with [3H]thymidine. Clone D7/64 cells were grown for 3 days in [3H]thymidine and half the cells were treated with TPA (1.6 X 10-7 M). The total number of cells in the cultures and the amount of trichloroacetic acid-insoluble radioactivity in 0.2-ml aliquots of cell suspension were determined each day thereafter. (O) Untreated cells; (0) TPA-treated cells.,of B+ cells. This increase was detected 2 days after removal and reached a peak 2-3 days later, irrespective of the length of time the cells had been treated with TPA (Fig. 4). In culltures that had been treated with TPA, the maximum percentage of B+ cells after its removal was approximately equal to the percentage of B+ cells in the clone at the beginning of the experiment (i.e., 60%). Thtis return to the original percentage-of different~iating cells was observed even after days of continuo'us exposure to TPA, at a time when cultures grown in medium without TPA had declined to a level of about 30% B+ cells (see Fig. 3). Follow-up of the cultures from which TPA had been removed showed that the differentiated phenotype was as unstable as in control cultures, with the percentage of B+ cells decreasing with time, according to the same kinetics as control cells. When the cells were reexposed to TPA, differentiation was again inhibited and to the same extent as in control cultures that had never been exposed to the promoter. We conclude that TPA trieatment can delay the reversion of spontaneously differentiating clones to a phenotype with fewer differentiating cells. Inhibition of Differentiation in Contrast to Selective Lysis of Differentiated Cells. The low percentage of B+ -cells in cultures treated with TPA could result either from inhibition of stem cell differentiation. or from selective lysis of B+ cells. z Proc. Natl. Acad. Sci. USA 74 (1977) The second explanation seems unlikely since long-term treatment with TPA appears to stabilize the spontaneously differentiating phenotype (see above), and this could only occur at the stem cell level. To further rule out the possibility that selective lysis of spontaneously differentiating cells is the mechanism of TPA action in this system, we did the following experiment. Cultures of clone D7/64 containing 42% B+ cells were labeled by growing them for 3 days in medium containing [3HIthymidine (0.1 ACi/ml; specific activity 6.7 Ci/mmol). The cultures were divided in two, and half the cells were treated with TPA. At daily intervals, the total number of cells in the cultures and the amount of trichloroacetic acid-insoluble radioactivity in 0.2-ml aliquots of cell suspension were determined. As shown in Fig. 5, the amount of acid-insoluble radioactivity at each time point was the same in TPA-treated as in control cultures and the amount per cell in both was diluted by cell division at the same rate. Since each B+ cell has the potential for up to five cell divisions,t a minimum decrease in acid-insoluble radioactivity of 8% would have been detected each day in this population that originally contained 42% B+ cells if selective lysis of B+ cells were operative. Effect of Tumor Promoters on Hemoglobin and Globin Chain Synthesis. To rule out the possibility that a decrease in benzidine reactivity reflects only a decrease in intracellular heme content rather than a decreased synthesis of hemoglobin, we determined the actual amounts of hemoglobin and globin chains synthesized in control and phorbol diester-treated cultures. The amount of hemoglobin was greatly reduced in TPA-treated and PDB-treated cultures compared to controls (Table 1). There was only a slight decrease in hemoglobin in PDA-treated cultures. Separation of denatured cytoplasmic proteins showed that a- and fl-globin chains were synthesized in both control and treated cultures (Fig. 6). The amounts were greatest in untreated cultures and least in TPA-treated cultures. These observations on hemoglobin and globin chain synthesis complement the findings evaluated by the benzidine reaction and support the interpretation that phorbol diesters with tumorpromoting activity restrict the expression of a product of differentiation, hemoglobin. DISCUSSION Within the series of phorbol diesters, TPA and PDD are the most active in promoting tumors on mouse skin. PDB is less active and PDA and 4-a-PDD are inactive, as is phorbol itself (5-7). As described in this paper, these compounds inhibited spontaneous differentiation of Friend erythroleukemia cells in culture with the same order of activity. Most striking is the Table 1. Effect of phorbol diesters on hemoglobin synthesis Phorbol Cells/ml % B+ Hemoglobin Proteint Clone diester* (X 106) cells (4g/106 cells) (,ug/106 cells) D7/64 TPA N.D. PDA N.D. PDB N.D. None N.D. D7/1 TPA PDA None Clone D7/64 cells were treated with the phorbol diesters for 6 days. Clone D7/1 cells were treated for 8 days with refeeding of the cultures after 4 days. The values shown are the averages from duplicate flasks. N.D. = not determined. * 1.6 X 10-7 M. t In the cytoplasmic lysate.

4 Cell Biology: Rovera et al. i'. a~~~~~~~~~~~~~. '4 **, wmd* 4. qf. _ _ I,.>,t... Jo And vw. hi _ ' 1X % _ t.s A. s.'s i _;. _w. t'' E.:,4;:, :.tp.:t * Hi..... 'I., :e>. Us... *r *. as ;:: *; A.. :w.. ',... s1 us*: I_ so _ A. i., item.: A:. Proc. Nati. Acad. Sci. USA 74 (1977) 2897,:.: _ LV- fjv 1J f J FIG. 6. Slab gel electrophoretic separation of globin chains. (Left) Autoradiogram; (Right) densitometer scan at 570 nm. The cells were grown for 8 days (two passages) in the absence or presence of the phorbol esters with a 14C-labeled amino acid mixture present in the medium (0.5 MCi/ml) for the last 96 hr. After fluorographic treatment, the gel was exposed for 4 days. All samples contained the same amounts of radioactivity per cell and the same amounts of protein were overlaid on the gels. The arrows indicate the a- and j3-globin chains that were identified by coelectrophoresis of unlabeled, purified DBA/2 mouse hemoglobin. fact that PDD is a potent tumor promoter in mouse skin and a potent inhibitor of differentiation in culture while the epimer, 4-a-PDD, is completely inactive in both systems. Inhibition of differentiation occurred at nontoxic concentrations of the active diesters. Inhibition by TPA was reversible; after removal of the compound, TPA-treated cells retained their potential for producing a high percentage of spontaneously differentiating cells, unlike control cells, which gradually reverted to a population with a low percentage of B+ cells. Weinstein et al. (9) have reviewed the many effects of tumor-promoting compounds on cellular physiology and biochemistry in culture. Some of the more recent observations made in different cell types are that TPA can induce plasminogen activator (19) and ornithine decarboxylase (), alter cell surface glycopeptides (9), and enhance saturation density (21). A model of tumor promotion in vitro has been described in which cell transformation occurs when a mouse embryo cell line, treated with nontransforming doses of ultraviolet radiation or 7,12-dimethylbenz[a]anthracene, is exposed to TPA (22, 23). An enhancing effect of TPA on the recovery of spontaneous and induced mutations from V79 Chinese hamster cells also has been reported recently (24). Here we report that phorbol diesters with tumor-promoting activity in mouse skin inhibit cell differentiation in culture. The inhibitory effect on differentiation apparently is not unique to the Friend erythroleukemia cell system. We have obtained preliminary evidence that TPA inhibits differentiation of mouse adipocytes and teratocarcinoma cells in culture. It is not yet known if the inhibition of differentiation by phorbol diester tumor promoters is directly related to any of the cellular effects so far ascribed to these compounds. If further testing should continue to show a direct correlation between known tumor-promoting activity in vivo and inhibition of differentiation in the Friend cell system, the system would be excellent for screening compounds for tumor-promoting activity and for studying the molecular basis of promotion. The cells grow well in suspension, they clone with high efficiency, and the product of differentiation is a well-characterized protein. One limitation may be that some of the compounds being screened will be cytotoxic (see ref. 25). The finding that active tumor promoters can inhibit cell differentiation suggests a possible mechanism for promotion. A shift of the cell population, under the influence of tumorpromoting agents, from a commitment to differentiate- (and become end cells) to a commitment to proliferate (and increase the stem cell population) may be the mechanism by which latent tumor cells can be activated or can acquire additional time to express the malignant phenotype before becoming end cells. Note Added in Proof. After this manuscript was submitted, a paper by Cohen et al. (26) described reversible inhibition by TPA of myogenesis in chick embryo muscle cultures. We acknowledge the excellent technical assistance of Christine Marsh and Janet Horn and we thank Dr. W. M. Baird for his advice. This work was supported by Research Grants CA10815, CA19454, and CA08936 from the National Cancer Institute. G.R. is a Scholar of the Leukemia Society of America. T.G.O'B. is a U.S. Public Health Service Post-Doctoral Fellow. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact. 1. Patuleia, M. C. & Friend, C. F. (1967) Cancer Res. 27, Friend, C., Preisler, H. D. & Scher, W. (1974) Curr. Top. Dev. Biol. 8, Harrison, P. R. (1976) Nature 262, Rovera, G. & Bonaiuto, J. (1976) Cancer Res. 36, Thielmann, H. W. & Hecker, E. (1969) in Fortschritte der

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