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[ 16] R e t i n o i d - B i n d i n g P r o t e i n s in E m b r y o n a l Carcinoma Cells B y JOSEPH F. GRIPPO a n d MICHAEL I. SHERMAN
Introduction The mechanism by which retinoids promote cell differentiation has yet to be elucidated, but current evidence suggests that retinoids alter the transcription of regulatory genes to initiate a cascade of events leading to the differentiated phenotype. By analogy with the steroid hormones, it has been proposed that retinoids interact with a receptor which can bind both its ligand and specific regulatory regions of the genome to modulate gene expression at the level of transcription.1 Considerable effort has gone into characterizing the proteins or receptors which presumably mediate retinoid action. Most research has centered on two retinoid-binding proteins, namely, the cellular retinoic acid-binding protein (CRABP) and the cellular retinol-binding protein (CRBP). Both CRABP and CRBP belong to a family of low molecular weight (15,000) proteins with eight known members, including CRBP II, three fatty acid-binding proteins, an adipocyte protein (aP2), and the P2 protein of peripheral nerve myelin.2,3 CRABP and CRBP exhibit high affinity, as well as selectivity, for their respective ligands, with apparent dissociation constants in the range of 10-20 nM. 4-7 Whereas specific nuclear binding sites for CRABP and CRBP appear to exist (see Ref. 8), data from Chytil's laboratory suggest that both CRABP and CRBP deliver their ligands to nuclear acceptor sites, but do not appear to be retained in the nuclear compartment. 9,1° In keeping with this observation, primary sequence data F. Chytil and D. Ong, Fed. Proc., Fed. Am. Soc. Exp. Biol. 38, 2510 (1979). 2 L. A. Demmer, E. H. Birkenmeier, D. A. Sweetser, M. S. Levin, S. Zollman, R. S. Sparkes, T. Mohandas, A. J. Lusis, and J. I. Gordon, J. Biol. Chem. 262, 2458 (1987). 3 j. Sundelin, S. R. Das, U. Eriksson, L. Rask, and P. A. Peterson, J. Biol. Chem. 260, 6494 (1985). 4 D. E. Ong and F. Chyfil, J. Biol. Chem. 250, 6113 (1975). s D. E. Ong and F. Chytil, J. Biol. Chem. 253, 828 (1978). 6 A. M. Jet'ten and M. E. R. Jetten, Nature (London) 278, 180 (1979). J. F. Gnppo and L. J. Gudas, or. Biol. Chem. 262, 4492 (1987). s M. I. Sherman, in "Retinoids and Cell Differentiation" (M. I. Sherman, ed.), p. 161. CRC Press, Boca Raton, Horida, 1986. 9 S. Takase, D. E. Ong, and F. Chytil, Proc. Natl. Acad. Sci. U.S,A. 76, 2204 (1979). ~oS. Takase, D. E. Ong, and F. Chytil, Arch. Biochem. Biophys. 247, 328 (1986).
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for both bovine adrenal CRABP 3 and rat liver CRBP n do not suggest the presence of DNA-binding domains. The recent discovery of nuclear retinoic acid receptors (RAR), which resemble the steroid receptor superfamily of transcriptional regulatory proteins, t2-~5 suggests that these are the proteins which directly modulate the effects of retinoids (or at least retinoic acids) on gene expression. This in turn provides a plausible role for the cellular retinoid-binding proteins, namely, to shuttle retinoids from the cytoplasm to the nucleus, where they interact with the RAR. Members of this class of proteins include RARot, 12't3 RARfl, 14 and the newly described RARe,. tSa They are 50-kDa proteins containing both ligand-binding and DNA-binding domains. As yet, no nuclear receptor specific for retinol binding has been described. Methods to characterize R A R are presented elsewhere) 6 This chapter focuses on procedures that can be employed in the characterization of the role of CRABP and CRBP in retinoid-related cellular behavior. Embryonal carcinoma (EC) cells are described here as prototypes for such studies because they, like m a n y other cell types, possess CRABP, CRBP, and RAR. Embryonal carcinoma cells differentiate readily in response to retinoids, an event which can be monitored in a number of convenient ways) 7 Furthermore, EC lines can be induced to metabolize retinoids, Is a phenomenon which might involve the retinoid-binding proteins) 9 We examine below methods to characterize CRABP and CRBP protein and RNA levels, and we also outline strategies for producing mutant cell lines that lack detectable CRABP activity.
II j. Sundelin, H. Anundi, L. Tragardh, U. Eriksson,P. Lind, H. Ronne, P. A. Peterson, and L. Rask, J. Biol. Chem. 260, 6488 (1985). t2 M. Petkovich,N. J. Brand, A. Krnst, and P. Chambon, Nature (London) 330, 444 (1987). 13V. Giguere, E. S. Ong, P. Segui,and R. M. Evans, Nature (London) 3311,624 (1987). 14N. Brand, M. Petkovich,A. Krust, P. Chambon, H. de The, A. Marchio, P. Tiollais,and A. Dejean, Nature (London) 332, 850 (1988). 15D. Benbrook, E. Lernhardt, and M. Pfahl, Nature (London) 333, 669 (1988). tsa A. Zelent, A. Krust, M. Petkovich, P. Kastner, and P. Chambon, Nature (London) 339, 714(1989). 16A. M. Jetten, J. F. GrilaPO,and C. Nervi, this seriesVol. 189 [25]. 17p. Abarzua and M. I. Sherman, this series, Vol. 189 [35]. 18M. L. Gubler and M. I. Sherman, this series, Vol. 189 [60]. 19M. L. Gubler and M. I. Sherman,J. Biol. Chem. 260, 9552 (1985).
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Cell Lines Numerous murine EC cell lines are available. 2° Two EC cell lines commonly used are F9 21,22 and PCC4.azalR. 23,24 Both F9 and PCC4.azalR cells are cultured in 5% CO2 at 37 ° in Dulbecco's modified Eagle's medium (DMEM), supplemented with 2 m M glutamine containing 10% heat-inactivated fetal calf serum. Under these conditions, the generation time of the cells is very short (10 hr or less). F9 cells can be grown in the same medium supplemented with 10% calf serum 7 (generation time 15- 18 hr). In either case, the cells fare best when grown on tissue culture plates coated with 0.15-0.3°/0 gelatin (see Ref. 17). Cell passage number should be recorded as cells should be discarded after 30-50 passages in order to avoid phenotypic drift. Typically, cells are plated at a density of 0.7 × 106/100-mm dish or 1.5 X l06 cells/150-mm dish and allowed to attach overnight.
Cytosol Preparation Approximately 5 × I0 s EC cellsgrowing in monolaycr (6- 15 150-mm dishes of confluent cells,depending on the celllineand the cultureconditions)arc washed with cold (Ca2+- and Mg2+-free phosphate-bufferedsaline (PBS) and removed from the platesin PBS with a rubber policeman or by trypsin treatment. 17 The cells are collected by centrifugation at 600 g for 5 rain at 4 ° and disrupted by Dounce homogenization (40 strokes) in 1 packed cell volume of homogenization buffer (10 raM) Tfis buffer, pH 7.4, 2 m M MgC12, and 7 m M 2-mercaptoethanol.6 Similar results are obtained when the MgC12 is omitted. 7 The cell homogenates are centrifuged at 100,000 g for 60 rain at 4 ° and stored in small aliquots at - 7 0 ° until assayed. Freezing and thawing do not seem to affect retinoid-binding activity. 25
2oG. R. Martin, in "Teratocarcinoma Stem Cells" (L. M. Silver, G. R. Martin, and S. Stdckland, eds.), p. 690. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1983. 21 E. G. Bernstine, M. L. Hooper, S, Grandchamp, and B. Ephrussi, Proc. Natl. Acad. Sci. U.S.A. 70, 3899 (1973). 22 S. Strickland and V. Mahdavi, Cell (Cambridge, Mass.) 15, 393 (1978). 23 H. Jakob, T. Boon, J. Galliard, J. F. Nicolas, and F. Jacob, Ann. Microbio[. Inst. Pasteur 124B, 269 (1973). 24A. M. Jetten, M. E. R. Jetten, and M. I. Sherman, Exp. CellRes. 124, 381 (1979). 25j. Schindler, K. I. Matthaei, and M. I. Sherman, Proc. Natl. Acad. Sci. U.S.A. 78, 1077 (1981).
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Cellular Retinoid-Binding A s s a y s Several methods are available for detecting CRABP and CRBP activity. These include sucrose gradient analyses, 7~5 dextran-treated charcol, 7 gel filtration and ion-exchange chromatography, 26-~ high-performance size-exclusion chromatography (HPSEC), 29 radioimmunoassay, and immunohistochemistry. 3°-u We describe below the sucrose gradient, dextran-charcoal, and Sephadex assays that we have used. In these procedures, retinoid-binding proteins are measured in high-speed cytosols. Attempts to measure this activity in particulate fractions are complicated by considerable levels of nonspecific binding. Even when cytosolic fractions are used, there should be a side-by-side control for each sample containing a 100- to 200-fold molar excess of unlabeled retinoic acid retinol in order to control for nonspecific binding. This applies for all of the procedures described below. Sucrose Gradient Analysis
Sucrose gradient analysis is the procedure used originally to characterize retinoid-binding protein activities. 4,5 It is accurate and effective but requires more time and cell extract protein than other techniques, and the availability of rotors and centrifuges limits the number of samples that can be analyzed simultaneously. At least 600/tg cytosol protein is mixed with 50 n M all-trans-[3H]retinoic acid (RA) for CRABP or [3H]retinol for CRBP activity determinations (both at 10-40 Ci/mmol) and adequate homogenization buffer to bring the total volume to 200/d. Ethanol is used to dissolve the retinoids in a volume not exceeding 4% of the total incubation volume. It should be stressed that inconsistent results are obtained with protein concentrations less than 2 - 3 mg/ml in this assay. 7 Samples are incubated for 3 - 5 hr at 4 °. After incubation, unbound ligand is removed by mixing with dextrancoated charcoal suspension (130/~1) followed by centrifugation. [Dextran26j. C. Saari, S. Futterman, G. W. Stubbs, J. T. Heffernan, L. Bredberg, K. Y. Chan, and D. W. Albert, Invest. Ophthalmol. Visual Sci. 17, 988 (1978). 27M. I. Sherman, M. L. Paternoster, and M. Taketo, Cancer Res. 43, 4283 (1983). 28U. Barkai and M. I. Sherman, J. CellBiol. 104, 671 (1987). 29H. E. Shubeita, M. D. Patel, and A. M. McCormick, Arch. Biochem. Biophys. 247, 280 (1986). 30M. Kato, K. Kato, W. S. Blaner, B. S. Chertow,and D. S. Goodman, Proc. Natl. Acad. Sci. U.S.A. 82, 2488 (1985). 31U. Eriksson, E. Hansson, M. Nilsson, K.-H. Jonsson, J. Sundelin, and P. A. Peterson, Cancer Res. 46, 717 (1986). 32M. Maden, D. E. Ong, D. Summerbell, and F. Chytil, Nature (London) 335, 733 (1988).
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coated charcoal is prepared by mixing 2.5% activated charcoal and 0.2% dextran sulfate (w/v) overnight at 4 °. Fines are removed by low-speed centrifugation, and the suspension is stored at 4°.] Two hundred fifty microliters of supernate is applied to a 5 - 20% sucrose gradient (5 ml) and spun at 220,000 g for 18 hr at 4 ° using a swinging-bucket rotor (SW50.1). After centrifugation, 280-/A aliquots (7-8 drops), withdrawn from the bottom of each tube, are collected into scintillation vials (5 ml cocktail) and counted in a liquid scintillation spectrometer. Myoglobin (2 S) and bovine serum albumin (4.6 S) can be used as sedimentation markers. CRABP and CRBP sediment with the 2 S marker. TM
Dextran-Coated Charcoal Assay In the dextran-coated charcoal assay, 450 #g (125/tl) of eytosol protein is incubated with [3H]retinoid in the assay mix described in the previous section. Unbound ligand is removed by mixing with 100/zl of dextrancoated charcoal suspension (prepared as described above), followed by centrifugation (3000 g, 20 min) to clarify the supernatant. As noted above, levels of nonspecific [3H]retinoid binding are determined by incubation of sample in the presence of a 100-fold molar excess of unlabeled retinoid. One hundred microliters of the clear supernatant is counted by liquid scintillation spectrometry, and specific [3H]retinoid binding, determined by subtracting nonspecific from total binding, is generally expressed as picomoles [3H]retinoid bound per milligram supernatant protein.
Sephadex Assays We have used two procedures involving Sephadex chromatography to measure retinoid-binding protein activities.27,2s Both are rapid and convenient and require only small amounts of cell extract for analysis of activity. The first assay27 combines the use of dextran-coated charcoal with Sephadex chromatography, which results in low background levels of radioactivity, thereby allowing the use of reduced amounts of cellular protein compared with the above procedures. The assay mixture is prepared and incubation carried out as described above except that only 100-250/tg cytosolic protein is added and the total volume is 150/11. To the assay mix is added 100/tl dextran-coated charcoal solution. After centrifugation (3000 g) for 20 min at 4 °, 100/A of supernatant is passed through a 7-cm Sephadex G-25-150 column in a Kimble Pasteur pipette (No. 72050). The bound counts (in the void volume) are eluted with 1 ml homogenization buffer and are collected directly into scintillation vials for radioactivity determinations. In an alternative procedure,2s the binding reaction is carried out on a disk from a Tetralute kit purchased from Ames-Yissum (Jerusalem, Israel);
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the disk is in turn placed on a 1 X 2 cm Sephadex G-25 column, which is used to separate bound from unbound [3H]retinoid after the incubation period has ended. Carrying out the incubation on the disk appears to facilitate the binding reaction, thus permitting shorter incubation times and the use of smaller amounts of radioactivity. This, in turn, obviates the need for the dextran-coated charcoal extraction step. Cytosol protein (50200/zg) is pipetted onto the disk followed by [3H]retinoic acid or [3H]retinol (3-4 × 105 cpm in 10/zl ethanol). Incubation buffer (25 m M Tris, pH 7.4, 25 m M NaC1) is added to give a total reaction volume of 300/zl. Control samples to determine nonspecific binding should contain no more than a 100-fold excess of the appropriate unlabeled retinoid. When [3H]retinol is used, 0.3 M sucrose is added to the incubation mix (in the absence of sucrose, spuriously high values for retinol binding are obtained for reasons that are unclear). After 90 rain at 4 °, the reaction mix is allowed to flow through the column followed by 500 #1 incubation buffer. Bound radioactive retinoid is then eluted into scintillation vials with 900 pl incubation buffer. All of these methods have been used successfully to determine levels of CRABP or CRBP. In each case, we have validated the assays carded out with F9, PCC4.azalR, and/or Nulli-SCC1 EC cells by comparing the results with those obtained simultaneously by sucrose gradient centrifugation; data obtained are in agreement at the level of within 10%. It must, however, be noted that neither the charcoal-dextran nor the Sephadex assay confirms that the retinoids are indeed bound to their respective binding proteins. Therefore, when these assays are being used for the first time, it would be prudent to validate them against the sucrose density gradient or HPSEC 29 assay. None of the techniques described above will physically separate CRABP from CRBP. These binding proteins can be distinguished pharmacologically, as a 200-fold molar excess of retinol will not displace [3H]retinoic acid binding from CRABP and a 200-fold molar excess of retinoic acid will not displace [3H]retinol binding from CRBP. Physical separation of the two proteins can be achieved by ion-exchange chromatography,u R N A Isolation and Northern Analysis
eDNA clones for CRABPaa,34 and for CRBP 35 have been obtained and have been used to characterize mRNA expression for these retinoid-bind33 H. E. Shubeita, J. F. Sambrook, and A. M. McCormick, Proc. Natl. Acad. Sci. U.S.A. 84, 5645 (1987). C. Stoner and L. J. Gudas, CancerRes. 49, 1497 (1989). 35 V. Colantuoni, R. Cortcs¢, M. Nilsson, J. Lundvall, C. Bavik, U. Eriksson, P. A. Peterson, and J. Sund¢lin, Biochem. Biophys. Res. Commun. 130, 431 (1985).
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ing proteins in F9 EC cells. 36 We have routinely used a guanidine-HC1 method to isolate RNA from monolayer cultures of EC cells. Confluent dishes of EC cells are rinsed with PBS and scraped into ice-cold 7M guanidine-HC1, 20 m M potassium acetate, pH 7.0, 5 m M EDTA, and 1 m M dithiothreitol and precipitated twice at - 2 0 ° from this solution by addition of one-half volume of ethanol. Following centdfugation the pellet is redissolved in 50 m M Tris-HC1, pH 9.0, 5 m M EDTA, 100 m M NaCl, and 0.5% sodium dodecyl sulfate and extracted twice with an equal volume of phenol/chloroform/isoamyl alcohol (10:9.6:0.4, v/v). RNA is then precipitated with 0.2 M NaCl and 2.5 volumes ethanol. RNA blot analysis is performed by standard procedures. RNA is fractionated by electrophoresis in 1% agarose/2.2 M formaldehyde slab gels, stained with ethidium bromide, transferred to nitrocellulose or nylon membranes, and attached to the filters by baking at 80 ° in vacuo. Filters are prehybridized in a suitable hybridization mixture 33 and hybridized with at least 106 cpm/ml labeled eDNA probe. When harvesting more than 3 X l07 cells, we use approximately 0.5 ml guanidine-HCl/10 ~ cells. Poly(A)+ RNA can be selected, but 15-30/lg total F9 EC RNA can be used to measure RNA levels for CRABP and CRBP. Both mouse CRABP and human CRBP eDNA probes hybridize with a low abundance, single mRNA species in F9 cells of about 800 nucleotides (CRABP mRNA in human Y79 retinoblastoma cells is slightly larger, 1000 nucleotides).37 Isolation of E m b r y o n a l Carcinoma Cell Lines Lacking Cellular Retinoic Acid-Binding Protein Activity The methods described above can be used to characterize the specific retinoid-binding capacity and expression of retinoid-binding protein mRNA species in EC cells. As one means of investigating the role of CRABP in mediating retinoid effects, mutant EC cell lines that lack functional CRABP activity can be isolated (the role, if any, of CRBP in retinoid-induced EC cell differentiation has yet to be elucidated, s and the isolation of mutant cells devoid of CRBP has not been reported). Resistance to retinoic acid-induced differentiation can be used as a convenient selection procedure for obtaining these lines because differentiated cells grow poorly, if at all, at clonal density. ~7 Two independent efforts have resulted in generation of CRABP-deficient EC cell lines) 5,37 In the first, PCC4.azalR cells were mutagenized by 36 S.-Y. Wang, G. J. LaRosa, and L. J. Gudas, Dev. Biol. 107, 75 (1985). 37 S.-Y. Wang and L. J. Gudas, J. Biol. Chem. 259, 5899 (1984).
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exposure to N-methyl-N'-nitronitrosoguanidine (MNNG) at 3 gg/ml for 18 hr followed by plating at clonal density. Retinoic acid-refractory cells were selected for clonal growth in l0 g M retinoic acid. Approximately 1 in 105 cells formed clones with an undifferentiated, EC-like appearance. Cells from these clones were passaged several times in the presence of l0 gM retinoic acid and were finally subcloned in the same retinoic acid-containing medium. Two differentiation-defective clonal EC cell lines, PCC4(RA)- 1 and PCC4(RA)- 2, which contain low or undetectable levels of CRABP activity, have been isolated in this way. The F9 RA3-10 line derived from F9 cells was obtained36by treating F9 wild-type cells (1-2 X 107 cells per T150 flask) with 5 gg/ml MNNG for 5 hr. Surviving cells were grown under nonselective conditions for 5 days and were then treated with 1 # M retinoic acid for 24 hr. The cells were removed from the flask with trypsin and cloned on plates containing 0.34% agarose over a mouse embryo fibroblast feeder layer in the presence of retinoic acid. This selection procedure is based on the observation that wild-type F9 cells will form colonies in soft agar whereas retinoic acidtreated differentiated derivatives will not. Colonies picked from these plates were expanded for further analysis. The F9 RA3-10 line isolated in this manner was found to lack mRNA and binding capacity for CRABP and also failed to differentiate in response to retinoic acid. It should be noted that these procedures can also result in the generation of differentiation-defective EC cells which nevertheless possess CRABP. 36,3s Therefore, any differentiation-defective lines which are obtained should be analyzed to identify those lacking CRABP mRNA and/ or binding activity. 38 p. A. McCue, K. I. Matthaei, M. Taketo, and M. I. Sherman, Dev. Biol. 96, 416 (1983).
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[ 16] R e t i n o i d - B i n d i n g P r o t e i n s in E m b r y o n a l Carcinoma Cells B y JOSEPH F. GRIPPO a n d MICHAEL I. SHERMAN
Introduction The mechanism by which retinoids promote cell differentiation has yet to be elucidated, but current evidence suggests that retinoids alter the transcription of regulatory genes to initiate a cascade of events leading to the differentiated phenotype. By analogy with the steroid hormones, it has been proposed that retinoids interact with a receptor which can bind both its ligand and specific regulatory regions of the genome to modulate gene expression at the level of transcription.1 Considerable effort has gone into characterizing the proteins or receptors which presumably mediate retinoid action. Most research has centered on two retinoid-binding proteins, namely, the cellular retinoic acid-binding protein (CRABP) and the cellular retinol-binding protein (CRBP). Both CRABP and CRBP belong to a family of low molecular weight (15,000) proteins with eight known members, including CRBP II, three fatty acid-binding proteins, an adipocyte protein (aP2), and the P2 protein of peripheral nerve myelin.2,3 CRABP and CRBP exhibit high affinity, as well as selectivity, for their respective ligands, with apparent dissociation constants in the range of 10-20 nM. 4-7 Whereas specific nuclear binding sites for CRABP and CRBP appear to exist (see Ref. 8), data from Chytil's laboratory suggest that both CRABP and CRBP deliver their ligands to nuclear acceptor sites, but do not appear to be retained in the nuclear compartment. 9,1° In keeping with this observation, primary sequence data F. Chytil and D. Ong, Fed. Proc., Fed. Am. Soc. Exp. Biol. 38, 2510 (1979). 2 L. A. Demmer, E. H. Birkenmeier, D. A. Sweetser, M. S. Levin, S. Zollman, R. S. Sparkes, T. Mohandas, A. J. Lusis, and J. I. Gordon, J. Biol. Chem. 262, 2458 (1987). 3 j. Sundelin, S. R. Das, U. Eriksson, L. Rask, and P. A. Peterson, J. Biol. Chem. 260, 6494 (1985). 4 D. E. Ong and F. Chyfil, J. Biol. Chem. 250, 6113 (1975). s D. E. Ong and F. Chytil, J. Biol. Chem. 253, 828 (1978). 6 A. M. Jet'ten and M. E. R. Jetten, Nature (London) 278, 180 (1979). J. F. Gnppo and L. J. Gudas, or. Biol. Chem. 262, 4492 (1987). s M. I. Sherman, in "Retinoids and Cell Differentiation" (M. I. Sherman, ed.), p. 161. CRC Press, Boca Raton, Horida, 1986. 9 S. Takase, D. E. Ong, and F. Chytil, Proc. Natl. Acad. Sci. U.S,A. 76, 2204 (1979). ~oS. Takase, D. E. Ong, and F. Chytil, Arch. Biochem. Biophys. 247, 328 (1986).
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for both bovine adrenal CRABP 3 and rat liver CRBP n do not suggest the presence of DNA-binding domains. The recent discovery of nuclear retinoic acid receptors (RAR), which resemble the steroid receptor superfamily of transcriptional regulatory proteins, t2-~5 suggests that these are the proteins which directly modulate the effects of retinoids (or at least retinoic acids) on gene expression. This in turn provides a plausible role for the cellular retinoid-binding proteins, namely, to shuttle retinoids from the cytoplasm to the nucleus, where they interact with the RAR. Members of this class of proteins include RARot, 12't3 RARfl, 14 and the newly described RARe,. tSa They are 50-kDa proteins containing both ligand-binding and DNA-binding domains. As yet, no nuclear receptor specific for retinol binding has been described. Methods to characterize R A R are presented elsewhere) 6 This chapter focuses on procedures that can be employed in the characterization of the role of CRABP and CRBP in retinoid-related cellular behavior. Embryonal carcinoma (EC) cells are described here as prototypes for such studies because they, like m a n y other cell types, possess CRABP, CRBP, and RAR. Embryonal carcinoma cells differentiate readily in response to retinoids, an event which can be monitored in a number of convenient ways) 7 Furthermore, EC lines can be induced to metabolize retinoids, Is a phenomenon which might involve the retinoid-binding proteins) 9 We examine below methods to characterize CRABP and CRBP protein and RNA levels, and we also outline strategies for producing mutant cell lines that lack detectable CRABP activity.
II j. Sundelin, H. Anundi, L. Tragardh, U. Eriksson,P. Lind, H. Ronne, P. A. Peterson, and L. Rask, J. Biol. Chem. 260, 6488 (1985). t2 M. Petkovich,N. J. Brand, A. Krnst, and P. Chambon, Nature (London) 330, 444 (1987). 13V. Giguere, E. S. Ong, P. Segui,and R. M. Evans, Nature (London) 3311,624 (1987). 14N. Brand, M. Petkovich,A. Krust, P. Chambon, H. de The, A. Marchio, P. Tiollais,and A. Dejean, Nature (London) 332, 850 (1988). 15D. Benbrook, E. Lernhardt, and M. Pfahl, Nature (London) 333, 669 (1988). tsa A. Zelent, A. Krust, M. Petkovich, P. Kastner, and P. Chambon, Nature (London) 339, 714(1989). 16A. M. Jetten, J. F. GrilaPO,and C. Nervi, this seriesVol. 189 [25]. 17p. Abarzua and M. I. Sherman, this series, Vol. 189 [35]. 18M. L. Gubler and M. I. Sherman, this series, Vol. 189 [60]. 19M. L. Gubler and M. I. Sherman,J. Biol. Chem. 260, 9552 (1985).
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Cell Lines Numerous murine EC cell lines are available. 2° Two EC cell lines commonly used are F9 21,22 and PCC4.azalR. 23,24 Both F9 and PCC4.azalR cells are cultured in 5% CO2 at 37 ° in Dulbecco's modified Eagle's medium (DMEM), supplemented with 2 m M glutamine containing 10% heat-inactivated fetal calf serum. Under these conditions, the generation time of the cells is very short (10 hr or less). F9 cells can be grown in the same medium supplemented with 10% calf serum 7 (generation time 15- 18 hr). In either case, the cells fare best when grown on tissue culture plates coated with 0.15-0.3°/0 gelatin (see Ref. 17). Cell passage number should be recorded as cells should be discarded after 30-50 passages in order to avoid phenotypic drift. Typically, cells are plated at a density of 0.7 × 106/100-mm dish or 1.5 X l06 cells/150-mm dish and allowed to attach overnight.
Cytosol Preparation Approximately 5 × I0 s EC cellsgrowing in monolaycr (6- 15 150-mm dishes of confluent cells,depending on the celllineand the cultureconditions)arc washed with cold (Ca2+- and Mg2+-free phosphate-bufferedsaline (PBS) and removed from the platesin PBS with a rubber policeman or by trypsin treatment. 17 The cells are collected by centrifugation at 600 g for 5 rain at 4 ° and disrupted by Dounce homogenization (40 strokes) in 1 packed cell volume of homogenization buffer (10 raM) Tfis buffer, pH 7.4, 2 m M MgC12, and 7 m M 2-mercaptoethanol.6 Similar results are obtained when the MgC12 is omitted. 7 The cell homogenates are centrifuged at 100,000 g for 60 rain at 4 ° and stored in small aliquots at - 7 0 ° until assayed. Freezing and thawing do not seem to affect retinoid-binding activity. 25
2oG. R. Martin, in "Teratocarcinoma Stem Cells" (L. M. Silver, G. R. Martin, and S. Stdckland, eds.), p. 690. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1983. 21 E. G. Bernstine, M. L. Hooper, S, Grandchamp, and B. Ephrussi, Proc. Natl. Acad. Sci. U.S.A. 70, 3899 (1973). 22 S. Strickland and V. Mahdavi, Cell (Cambridge, Mass.) 15, 393 (1978). 23 H. Jakob, T. Boon, J. Galliard, J. F. Nicolas, and F. Jacob, Ann. Microbio[. Inst. Pasteur 124B, 269 (1973). 24A. M. Jetten, M. E. R. Jetten, and M. I. Sherman, Exp. CellRes. 124, 381 (1979). 25j. Schindler, K. I. Matthaei, and M. I. Sherman, Proc. Natl. Acad. Sci. U.S.A. 78, 1077 (1981).
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Cellular Retinoid-Binding A s s a y s Several methods are available for detecting CRABP and CRBP activity. These include sucrose gradient analyses, 7~5 dextran-treated charcol, 7 gel filtration and ion-exchange chromatography, 26-~ high-performance size-exclusion chromatography (HPSEC), 29 radioimmunoassay, and immunohistochemistry. 3°-u We describe below the sucrose gradient, dextran-charcoal, and Sephadex assays that we have used. In these procedures, retinoid-binding proteins are measured in high-speed cytosols. Attempts to measure this activity in particulate fractions are complicated by considerable levels of nonspecific binding. Even when cytosolic fractions are used, there should be a side-by-side control for each sample containing a 100- to 200-fold molar excess of unlabeled retinoic acid retinol in order to control for nonspecific binding. This applies for all of the procedures described below. Sucrose Gradient Analysis
Sucrose gradient analysis is the procedure used originally to characterize retinoid-binding protein activities. 4,5 It is accurate and effective but requires more time and cell extract protein than other techniques, and the availability of rotors and centrifuges limits the number of samples that can be analyzed simultaneously. At least 600/tg cytosol protein is mixed with 50 n M all-trans-[3H]retinoic acid (RA) for CRABP or [3H]retinol for CRBP activity determinations (both at 10-40 Ci/mmol) and adequate homogenization buffer to bring the total volume to 200/d. Ethanol is used to dissolve the retinoids in a volume not exceeding 4% of the total incubation volume. It should be stressed that inconsistent results are obtained with protein concentrations less than 2 - 3 mg/ml in this assay. 7 Samples are incubated for 3 - 5 hr at 4 °. After incubation, unbound ligand is removed by mixing with dextrancoated charcoal suspension (130/~1) followed by centrifugation. [Dextran26j. C. Saari, S. Futterman, G. W. Stubbs, J. T. Heffernan, L. Bredberg, K. Y. Chan, and D. W. Albert, Invest. Ophthalmol. Visual Sci. 17, 988 (1978). 27M. I. Sherman, M. L. Paternoster, and M. Taketo, Cancer Res. 43, 4283 (1983). 28U. Barkai and M. I. Sherman, J. CellBiol. 104, 671 (1987). 29H. E. Shubeita, M. D. Patel, and A. M. McCormick, Arch. Biochem. Biophys. 247, 280 (1986). 30M. Kato, K. Kato, W. S. Blaner, B. S. Chertow,and D. S. Goodman, Proc. Natl. Acad. Sci. U.S.A. 82, 2488 (1985). 31U. Eriksson, E. Hansson, M. Nilsson, K.-H. Jonsson, J. Sundelin, and P. A. Peterson, Cancer Res. 46, 717 (1986). 32M. Maden, D. E. Ong, D. Summerbell, and F. Chytil, Nature (London) 335, 733 (1988).
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coated charcoal is prepared by mixing 2.5% activated charcoal and 0.2% dextran sulfate (w/v) overnight at 4 °. Fines are removed by low-speed centrifugation, and the suspension is stored at 4°.] Two hundred fifty microliters of supernate is applied to a 5 - 20% sucrose gradient (5 ml) and spun at 220,000 g for 18 hr at 4 ° using a swinging-bucket rotor (SW50.1). After centrifugation, 280-/A aliquots (7-8 drops), withdrawn from the bottom of each tube, are collected into scintillation vials (5 ml cocktail) and counted in a liquid scintillation spectrometer. Myoglobin (2 S) and bovine serum albumin (4.6 S) can be used as sedimentation markers. CRABP and CRBP sediment with the 2 S marker. TM
Dextran-Coated Charcoal Assay In the dextran-coated charcoal assay, 450 #g (125/tl) of eytosol protein is incubated with [3H]retinoid in the assay mix described in the previous section. Unbound ligand is removed by mixing with 100/zl of dextrancoated charcoal suspension (prepared as described above), followed by centrifugation (3000 g, 20 min) to clarify the supernatant. As noted above, levels of nonspecific [3H]retinoid binding are determined by incubation of sample in the presence of a 100-fold molar excess of unlabeled retinoid. One hundred microliters of the clear supernatant is counted by liquid scintillation spectrometry, and specific [3H]retinoid binding, determined by subtracting nonspecific from total binding, is generally expressed as picomoles [3H]retinoid bound per milligram supernatant protein.
Sephadex Assays We have used two procedures involving Sephadex chromatography to measure retinoid-binding protein activities.27,2s Both are rapid and convenient and require only small amounts of cell extract for analysis of activity. The first assay27 combines the use of dextran-coated charcoal with Sephadex chromatography, which results in low background levels of radioactivity, thereby allowing the use of reduced amounts of cellular protein compared with the above procedures. The assay mixture is prepared and incubation carried out as described above except that only 100-250/tg cytosolic protein is added and the total volume is 150/11. To the assay mix is added 100/tl dextran-coated charcoal solution. After centrifugation (3000 g) for 20 min at 4 °, 100/A of supernatant is passed through a 7-cm Sephadex G-25-150 column in a Kimble Pasteur pipette (No. 72050). The bound counts (in the void volume) are eluted with 1 ml homogenization buffer and are collected directly into scintillation vials for radioactivity determinations. In an alternative procedure,2s the binding reaction is carried out on a disk from a Tetralute kit purchased from Ames-Yissum (Jerusalem, Israel);
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the disk is in turn placed on a 1 X 2 cm Sephadex G-25 column, which is used to separate bound from unbound [3H]retinoid after the incubation period has ended. Carrying out the incubation on the disk appears to facilitate the binding reaction, thus permitting shorter incubation times and the use of smaller amounts of radioactivity. This, in turn, obviates the need for the dextran-coated charcoal extraction step. Cytosol protein (50200/zg) is pipetted onto the disk followed by [3H]retinoic acid or [3H]retinol (3-4 × 105 cpm in 10/zl ethanol). Incubation buffer (25 m M Tris, pH 7.4, 25 m M NaC1) is added to give a total reaction volume of 300/zl. Control samples to determine nonspecific binding should contain no more than a 100-fold excess of the appropriate unlabeled retinoid. When [3H]retinol is used, 0.3 M sucrose is added to the incubation mix (in the absence of sucrose, spuriously high values for retinol binding are obtained for reasons that are unclear). After 90 rain at 4 °, the reaction mix is allowed to flow through the column followed by 500 #1 incubation buffer. Bound radioactive retinoid is then eluted into scintillation vials with 900 pl incubation buffer. All of these methods have been used successfully to determine levels of CRABP or CRBP. In each case, we have validated the assays carded out with F9, PCC4.azalR, and/or Nulli-SCC1 EC cells by comparing the results with those obtained simultaneously by sucrose gradient centrifugation; data obtained are in agreement at the level of within 10%. It must, however, be noted that neither the charcoal-dextran nor the Sephadex assay confirms that the retinoids are indeed bound to their respective binding proteins. Therefore, when these assays are being used for the first time, it would be prudent to validate them against the sucrose density gradient or HPSEC 29 assay. None of the techniques described above will physically separate CRABP from CRBP. These binding proteins can be distinguished pharmacologically, as a 200-fold molar excess of retinol will not displace [3H]retinoic acid binding from CRABP and a 200-fold molar excess of retinoic acid will not displace [3H]retinol binding from CRBP. Physical separation of the two proteins can be achieved by ion-exchange chromatography,u R N A Isolation and Northern Analysis
eDNA clones for CRABPaa,34 and for CRBP 35 have been obtained and have been used to characterize mRNA expression for these retinoid-bind33 H. E. Shubeita, J. F. Sambrook, and A. M. McCormick, Proc. Natl. Acad. Sci. U.S.A. 84, 5645 (1987). C. Stoner and L. J. Gudas, CancerRes. 49, 1497 (1989). 35 V. Colantuoni, R. Cortcs¢, M. Nilsson, J. Lundvall, C. Bavik, U. Eriksson, P. A. Peterson, and J. Sund¢lin, Biochem. Biophys. Res. Commun. 130, 431 (1985).
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ing proteins in F9 EC cells. 36 We have routinely used a guanidine-HC1 method to isolate RNA from monolayer cultures of EC cells. Confluent dishes of EC cells are rinsed with PBS and scraped into ice-cold 7M guanidine-HC1, 20 m M potassium acetate, pH 7.0, 5 m M EDTA, and 1 m M dithiothreitol and precipitated twice at - 2 0 ° from this solution by addition of one-half volume of ethanol. Following centdfugation the pellet is redissolved in 50 m M Tris-HC1, pH 9.0, 5 m M EDTA, 100 m M NaCl, and 0.5% sodium dodecyl sulfate and extracted twice with an equal volume of phenol/chloroform/isoamyl alcohol (10:9.6:0.4, v/v). RNA is then precipitated with 0.2 M NaCl and 2.5 volumes ethanol. RNA blot analysis is performed by standard procedures. RNA is fractionated by electrophoresis in 1% agarose/2.2 M formaldehyde slab gels, stained with ethidium bromide, transferred to nitrocellulose or nylon membranes, and attached to the filters by baking at 80 ° in vacuo. Filters are prehybridized in a suitable hybridization mixture 33 and hybridized with at least 106 cpm/ml labeled eDNA probe. When harvesting more than 3 X l07 cells, we use approximately 0.5 ml guanidine-HCl/10 ~ cells. Poly(A)+ RNA can be selected, but 15-30/lg total F9 EC RNA can be used to measure RNA levels for CRABP and CRBP. Both mouse CRABP and human CRBP eDNA probes hybridize with a low abundance, single mRNA species in F9 cells of about 800 nucleotides (CRABP mRNA in human Y79 retinoblastoma cells is slightly larger, 1000 nucleotides).37 Isolation of E m b r y o n a l Carcinoma Cell Lines Lacking Cellular Retinoic Acid-Binding Protein Activity The methods described above can be used to characterize the specific retinoid-binding capacity and expression of retinoid-binding protein mRNA species in EC cells. As one means of investigating the role of CRABP in mediating retinoid effects, mutant EC cell lines that lack functional CRABP activity can be isolated (the role, if any, of CRBP in retinoid-induced EC cell differentiation has yet to be elucidated, s and the isolation of mutant cells devoid of CRBP has not been reported). Resistance to retinoic acid-induced differentiation can be used as a convenient selection procedure for obtaining these lines because differentiated cells grow poorly, if at all, at clonal density. ~7 Two independent efforts have resulted in generation of CRABP-deficient EC cell lines) 5,37 In the first, PCC4.azalR cells were mutagenized by 36 S.-Y. Wang, G. J. LaRosa, and L. J. Gudas, Dev. Biol. 107, 75 (1985). 37 S.-Y. Wang and L. J. Gudas, J. Biol. Chem. 259, 5899 (1984).
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exposure to N-methyl-N'-nitronitrosoguanidine (MNNG) at 3 gg/ml for 18 hr followed by plating at clonal density. Retinoic acid-refractory cells were selected for clonal growth in l0 g M retinoic acid. Approximately 1 in 105 cells formed clones with an undifferentiated, EC-like appearance. Cells from these clones were passaged several times in the presence of l0 gM retinoic acid and were finally subcloned in the same retinoic acid-containing medium. Two differentiation-defective clonal EC cell lines, PCC4(RA)- 1 and PCC4(RA)- 2, which contain low or undetectable levels of CRABP activity, have been isolated in this way. The F9 RA3-10 line derived from F9 cells was obtained36by treating F9 wild-type cells (1-2 X 107 cells per T150 flask) with 5 gg/ml MNNG for 5 hr. Surviving cells were grown under nonselective conditions for 5 days and were then treated with 1 # M retinoic acid for 24 hr. The cells were removed from the flask with trypsin and cloned on plates containing 0.34% agarose over a mouse embryo fibroblast feeder layer in the presence of retinoic acid. This selection procedure is based on the observation that wild-type F9 cells will form colonies in soft agar whereas retinoic acidtreated differentiated derivatives will not. Colonies picked from these plates were expanded for further analysis. The F9 RA3-10 line isolated in this manner was found to lack mRNA and binding capacity for CRABP and also failed to differentiate in response to retinoic acid. It should be noted that these procedures can also result in the generation of differentiation-defective EC cells which nevertheless possess CRABP. 36,3s Therefore, any differentiation-defective lines which are obtained should be analyzed to identify those lacking CRABP mRNA and/ or binding activity. 38 p. A. McCue, K. I. Matthaei, M. Taketo, and M. I. Sherman, Dev. Biol. 96, 416 (1983).
