Metabolism of Retinoids by Embryonal Carcinoma Cells*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The American Society of Biological Chemists, Inc. Val. 260, No. 17, Issue of August 15, pp ,1985 Printed in U. S. A. Metabolism of Retinoids by Embryonal Carcinoma Cells* (Received for publication, August 16, 1984, and in revised form, December 3, 1984) Mary Lou Gubler and Michael I. Sherman$ From the Department of Cell Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey Several embryonal carcinoma (EC) cell lines were tested in culture for their ability to metabolize alltr~ns-[~h]retinol, all-tr~ns-[~h]retinyl acetate, and all-tr~ns-[~h]retinoic acid. There was little, if any, metabolism of all-trans-retinol to more polar compounds; we failed to detect conversion to acidic retinoids by reverse-phase high performance liquid chromatography and derivatization. We also did not observe [3H]retinoic acid when EC cells were incubated with [ Hlretinyl acetate. Unlike the other retinoids, all-trans-[3h]retinoic acid, even at micromolar levels, was almost totally modified by cells from several EC lines within 24 h. Most of the labeled products were secreted into the medium. Some EC lines metabolized retinoic acid constitutively, whereas others had an inducible enzyme system. A differentiation-defective line, which contains little or no cellular retinoic acidbinding protein activity, metabolized retinoic acid poorly, even after exposure to inducers. At least eight retinoic acid metabolites were generated; many contain hydroxyl residues. Our data lead us to propose that retinol does not induce differentiation of EC cells in vitro via conversion to retinoic acid. Also, the relatively rapid metabolism of retinoic acid by EC cells suggests either that the induction of differentiation need involve only a transient exposure to this retinoid or that one or more of the retinoic acid metabolites can also promote differentiation. Of all known agents which induce differentiation of embryonal carcinoma (EC ) cells, retinoic acid is among the most potent and reliable. This retinoid induces differentiation of many cultured EC lines at concentrations within the nanomolar range (Strickland and Mahdavi, 1978; Jetten et al., 1979). Notable exceptions are a series of mutagenesis-derived, differentiation-defective EC lines which fail to respond to retinoic acid (Schindler et al., 1981; McCue et al., 1983; Wang and Gudas, 1984) and one line, PCC4.aza1, for which retinoic acid is toxic (Strickland and Mahdavi, 1978; Jetten et al., 1979). All-trans- and 13-cis-retinoic acids appear to be equally effective (Sherman et al., 1983a). Several retinoid metabolites and analogs have been tested for their ability to stimulate differentiation of EC cells (Strickland and Mahdavi, 1978; Jetten and Jetten, 1979; Trown et al., 1980; Sherman et al., 1983a); in general, retinoids which possess this activity are * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. The abbreviations used are: EC, embryonal carcinoma; CRABP, cellular retinoic acid-binding protein; HPLC, high performance liquid chromatography. capable of binding to a cellular retinoic acid-binding protein (CRABP). Retinol is exceptional in that it fails to bind to the CRABP, but is capable of inducing differentiation of several EC lines. However, higher concentrations of, and longer exposures to, retinol are often required to elicit an effect equivalent to retinoic acid (Eglitis and Sherman, 1983; Jetten and De Luca, 1983). Curiously, although PCC4.azal cells are killed by exposure to retinoic acid, they survive and differentiate in response to retinol (Eglitis and Sherman, 1983), whereas the PCC4.azalR subline (Jetten et al., 1979), which tolerates retinoic acid and differentiates in response to it, fails to differentiate upon exposure to relatively low levels of retinol and dies at higher concentrations (Eglitis and Sherman, 1983). The above observations and the findings that differentiation-defective EC cells which fail to respond to retinoic acid are also refractory to retinol (Eglitis and Sherman, 1983; Sherman et al., 1983b; Wang and Gudas, 1984) suggested to us that retinol might be converted to retinoic acid by cultured EC cells. Accordingly, we have tested this possibility with several EC lines. We have also studied the fate of all-transretinoic acid in EC cell cultures. EXPERIMENTAL PROCEDURES AND RESULTS? DISCUSSION At concentrations of retinol which promote differentiation of F9 cells (Eglitis and Sherman, 1983), esterification presumably occurs, and there might be some enzymatic isomerization to 13-cis-retinol and low levels of metabolism to two hydroxylated retinoids. However, we consistently failed to detect any conversion to retinal or retinoic acid during incubation periods as lengthy as 72 h. The same is true following preincubation of cells with retinoic acid (data not shown). We have estimated that retinol is 7-15% as effective as retinoic acid in promoting differentiation of F9 cells under our culture conditions (Eglitis and Sherman, 1983). From our derivatization analyses, we have failed to observe as little as 1% all-trans- t3h]retinoic acid or 13-~is-[~H]retinoic acid in extracts from either cells or their culture media. It is possible that some [3H]retinoic acid was synthesized but subsequently metabolized. If this were the case, however, the level of conversion would have to have been low since no differences were detected in the profiles of extracts derivatized with acetic anhydride alone or with diazomethane followed by acetic anhydride (Fig. 2, C and 8 ). It therefore appears unlikely that Portions of this paper (including Experimental Procedures, Results, Figs. 1-6, and Tables I and 11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD Request Document No. 84M-2567, cite the authors, and include a check or money order for $8.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. 9552

2 Retinoid Metabolism in Embryonal Carcinoma Cells 9553 retinol promotes differentiation of EC cells in vitro via me- considered it possible that retinoic acid is toxic to PCC4.azal tabolism to retinoic acid. Similar conclusions have been cells because they are unable to metabolize retinoic acid and reached by Williams and Napoli (1984), although supporting instead accumulate lethalevels of the retinoid. Instead, these data have not been published. cells were found to metabolize retinoic acid constitutively. In their abstract, Williams and Napoli (1984) reported that Conversely, we tested PCC4(RA)-2 cells for their ability to F9 cells esterify retinol and accumulate these metabolites. metabolize retinoic acid because we thought the lack of Although we have seen no evidence for conversion of [3H] CRABP in these cells might leave the retinoic acid unproretinol to retinyl acetate, the observation that 15-25% of the tected and thus vulnerable to the metabolic enzyme system, label is retained on the column under our routine high per- yet Table I illustrates that these cells metabolize retinoic acid formance liquid chromatography conditions and about half of inefficiently, even after pretreatment with retinoic acid or that material elutes with or near a retinyl palmitate standard retinol. The striking difference between PCC4(RA)-2 cells in 98% acetonitrile supports their contention. The lack of this and those of the parental PCC4.azalR line with respect to, material in the culture medium also confirms that the esteri- ability to metabolize retinoic acid raises the possibility that fied material is not secreted. The likelihood that at least some CRABP actually mediates this event, either by promoting [3H]retin~l is esterified suggests that our failure to detect [3H] enzyme synthesis or facilitating the interaction between reretinoic acid is not due to the inability of EC cells to take up tinoic acid and the enzymes that metabolize it (see, e.g. Saari the retinoid. and Bredberg, 1982). The data in Figs. 1 and 2 indicate that some of the polar If the metabolism of retinoic acid takes place in the microderivatives of [3H]retinol are either taken up poorly by EC somal compartment of EC cells, as is the case in hamster cells or secreted more efficiently than [3H]retinol. The same tissues (Roberts et al., 1979b; Roberts, 1981), our observations is true of [3H]retinyl acetate. However, after the latter com- pound is de-esterified by serum in the culture medium, the resulting [3H]retin~l is taken up by EC cells. Thereafter, its fate seems to be the same as that of [3H]retin~l added directly to the culture medium. Our inability to detect retinoic acid formation from retinyl acetate by EC cells contrasts with the studies of Frolik et al. (1981) in which hamster tracheal organ cultures produced low but detectable levels of retinoic acid from retinyl acetate. It is not clear whether this difference is due to cell type or organ versus cell culture. We have also failed to detect retinol conversion to retinoic acid by other cell lines, including cells derived from testes, a target organ for re ti no id^.^ Retinoic acid is metabolized extensively by F9 cells. This conclusion was also drawn by Williams and Napoli (1984). The retinoic acid was converted to several polar metabolites, as has been observed in animals (e.g. Frolik et al., 1978; Roberts et al., 1979a; Napoli and McCormick, 1981) and in tracheal explants (Frolik et al., 1978; Frolik, 1981). Retinoic acid induces its own metabolism in F9 cells. This is reminis- cent of the in uiuo observations of Roberts et al. (1979a, 1979b; Roberts, 1981): she and her colleagues have demonstrated that orally administered retinoic acid is sluggishly metabolized by vitamin A-deficient hamsters, whereas animals prefed retinoic acid metabolized subsequently administered [3H] retinoic acid extensively. It was shown further that microsomal extracts or tracheal explants from animals fed retinoic acid were substantially more active in metabolizing retinoic acid than were similar extracts or explants from hypovitaminotic A hamsters (Roberts et al., ). The present study extends these observations by demonstrating that the induction event can take place in culture. It is notable that induction with retinoic acid requires 2-3 days in vivo (Roberts et al., 1979b), but only a matter of hours in culture. Roberts et al. (1979b; Roberts, 1981) reported that retinoic acid metabolism was only inducible in some tissues (intestine, M. L. Gubler and M. I. Sherman, manuscript in preparation. show dramatically that, at least in culture, retinoic acid is readily accessible to EC cells. It is notable that within 5 h, induced F9 cells can metabolize micromolar amounts of retinoic acid. Since less than 5% of labeled [3H]retinoic acid (at either 10"j M or less than M) is ever cell-associated, our data suggest strongly that retinoic acid is taken up by EC cells, metabolized, and then relatively rapidly secreted. The results of pulse and pulse-chase experiments are consistent with this view.3 The effective secretion of the retinoic acid metabolites would, indeed, be expected if they are detoxification or deactivation products, as has been proposed (Roberts et al., 1979a; Roberts, 1981). We are currently testing whether retinoic acid is metabolized by microsomal enzymes in EC cells, as is the case for other tissues (Roberts et al., 1979a, 1979b). According to the scheme presented by Roberts (1981), retinoic acid in induced cells is first converted to 4-hydroxyretinoic acid followed by further oxidation to 4-oxo-retinoic acid and subsequently to other, more polar, metabolites. We have tentatively identified 4-hydroxy-retinoic acid as a retinoic acid metabolite in induced F9 cells, but we have failed to find significant amounts of labeled material coeluting with all-trans-4-oxo-retinoic acid following derivatization (Figs. 5 and 6). At least one polar metabolite of [3H]retinoic acid (peak 4 in Fig. 5H) does not appear to be hydroxylated, however. It could contain a keto or aldehyde residue other than in the 4 position. The major labeled polar metabolites (peaks 2 and 3 in Fig. 5H) are hydroxylated, although not further identified. These residues might occur in any, or a combination, of several positions on the retinoic acid molecule (see, e.g. Rietz et al., 1974). It will be important to determine whether the retinoic acid metabolites bind to CRABP and promote differentiation of EC cells. Roberts (1981) has indicated that several retinoic acid metabolites are biologically less active than retinoic acid liver); testes and tracheal microsomal extracts were consti- or inactive. If F9 metabolites are inactive in promoting differtutive for retinoic acid metabolism whereas kidney micro- entiation of these cells, then the metabolism of most added somal extracts, even those from retinoic acid-treated animals, retinoic acid within several hours would indicate that the appeared inactive. We have observed such differences among molecule is essential only for initiating the cascade of events the EC lines tested (Table I), as well as among other non-ec leading ultimately to overt differentiation of EC cells. cell lines3 The significance of the variation we observed is unclear, and it does not readily explain the different responses Acknowledgrnents-We are most grateful to A. Liebman, M. Roto retinoic acid of the EC lines tested. For example, we senberger, c. BuggB, c. Perry, w. Burger, D. Malarek, F. Vane, B. Pawson, and A. Shatkin for assistance, advice, synthesis of retinoids, and comments on the manuscript.

3 9554 Retinoid Metabolism REFERENCES Eglitis, M. E., and Sherman, M. I. (1983) Enp. Cell Res. 146, Frolik, C. A. (1981) Ann. N. Y. Acad. Sci. 359,37-44 Frolik, C. A., Tavela, T. E., Newton, D. L., and Sporn, M. B. (1978) J. Biol. Chem. 253, Frolik, C. A,, Dart, L. L., and Sporn, M. B. (1981) Biochim. Biophys. Acta 663, Jetten, A. M., and Jetten, M. E. R. (1979) Nature 278, Jetten, A. M., and De Luca, L.M. (1983) Biochem. Biophys. Res. Commun. 114, Jetten, A. M., Jetten, M. E. R., and Sherman, M. I. (1979) Exp. Cell Res. 124, McClean, S. W., Rudel, M. E., Gross, E. G., DeGiovanna, J. J., and Peck, G. L. (1982) Clin. Chem. 28, McCormick, A. M., Kroll, K. D., and Napoli, J. L. (1983) Biochemistry 22, McCue, P. A., Matthaei, K. I., Taketo, M., and Sherman, M. I. (1983) Deu. Biol. 96, NaDoli, J. L., and McCormick, A. M. (1981) Biochim. Biophys. Acta 666; Rietz, P., Wiss, O., and Weber, F. (1974) Vitam. Horm. 32, in Embryonal Carcinoma Cells Roberts, A. B. (1981) Ann. N. Y. Acad. Sci. 359,45-53 Roberts, A. B., Nichols, M.D., Newton, D. L., and Sporn, M.B. (1979a) J. Biol. Chem. 254, Roberts, A. B., Frolik, C. A., Nichols, M. D., and Sporn, M. B. (1979b) J. Biol. Chem. 254, Saari, J. C., and Bredberg, L. (1982) Biochim. Biophys. Acta 716, Schindler, J., Matthaei, K. I., and Sherman, M. I. (1981) Proc. Natl. Acad. Sci. U. S. A. 73, Sherman, M. I., Paternoster, M. L., and Taketo, M. (1983a) Cancer Res. 43, Sherman, M. I., Paternoster, M. L., Eglitis, M. A., and McCue, P. A. (1983b) in Teratocarcinoma Stem Cells (Silver, L., Martin, G. R., and Strickland, S., eds) p. 83, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Strickland, S., and Mahdavi, V. (1978) Cell 15, Trown, P. W., Palleroni, A. V., Bohoslawec, O., Richelo, B. N., Halpern, J. N., Gizzi, N., Lewinski, C., Machlin, L. J., Jetten, A., and Jetten, M. E. R. (1980) Cancer Res. 40, Vane, F. M,, BuggL., J. L., and Williams, T. H. (1982) Drug Metab. D~s~os. 10, Wang, S.-Y., and Gudas, L. J. (1984) J. Biol. Chem. 259, Williams, J. B., and Napoli, J. L. (1984) Fed. Proc. 43, 788 RESULTS

4 Retinoid Metabolism in Embryonal Carcinoma Cells IO TIME (mid

5 9556 Retinoid Metabolism in Embryonal Carcinoma Cells 5 IO 15 2ol IO

6 Retinoid Metabolism in Embryonal Carcinoma Cells 9557 k" a No tells F9 F9b oc15 51 PCC4.az.ilR 40 VCC4.dZdl PCC4(IA)P

7 9558 Retinoid Metabolism in Embryonal Carcinoma Cells 0 6? 20 IO 8 5 IO ?