J. Biol. Chem.-1983-Miller-3523-7

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					THE.JOURNAl.

OF BIOI.OGICAL CHEMISTRY Vol. 258, No. 6, Issue of March 25, pp. 3523-:152$, 1983 Printed an U.S.A.

Biochemical and GeneticAnalysis of Variant Mouse Hepatoma Cells Defective in the Induction Benzo(a)pyrene-metabolizing Enzyme of Activity*
(Received for publication, August 19, 1982)

Arthur G. Miller$, David Israel, and James P. Whitlock, Jr.8
From the Department of Pharmacology, Stanford University School of Medicine, Stanford, California94305

We have analyzed wild type mouse hepatoma (Hepa disappearance of BPI fluorescence from cells (10-12). In adl c l c l ) cells and variantcells which are defective the dition, the aryl hydrocarbon hydroxylase assay (13), which in induction of benzo(u)pyrene-metabolizing enzyme ac- measures the fluorescence of phenolic metabolites of BP, is a tivity. One type of variant has no detectable basal or sensitive method for studying mixed function oxidase activity inducible aryl hydrocarbon hydroxylase activity. This in cells in culture. Several polycyclic aromatic compounds are class contains apparently normal cytosolic receptors able to induce aryl hydrocarbon hydroxylase activity (7, 14). but for 2,3,7,8-tetrachlorodibenzo-p-dioxin, is unable to Studies using the most potent known inducer, 2,3,7,8-tetratranslocatetheinducer-receptorcomplex to the nuchlorodibenzo-p-dioxin, suggest that aryl hydrocarbon hycleus. The second type of variant low levels of basal droxylase induction is mediated by a cytosolic receptor prohas and inducible aryl hydrocarbon hydroxylase activity. tein, which binds the inducer and translocates it to the nuThis class contains cytosolic receptors which are de- cleus, leading to induction of enzyme activity (7, 15). creased either their number or their ability bind in in to We are studying the induction of the BP-metabolizing en2,3,7,8-tetrachlorodibenzo-p-dioxin; translocation of zyme system in mouse hepatoma (Hepa lclc7) cells in culture. the inducer-receptor complexto the nucleus is apparently normal. Cell fusions indicate that both variant Our approach has been to analyze variant cells which have defects in the induction response. Here, using both biochemphenotypesare recessive with respect to wild type. ical Complementationanalyses indicate that the defects are and genetic techniques, we characterize several variants which have defective aryl hydrocarbonhydroxylase induction located on different genes. mechanisms. Cell fusion analyses indicate that the variants are in different complementation groups. One type of variant, which contains low aryl hydrocarbon hydroxylase activity, Microsomal cytochrome P-450-containing mixed function has an altered cytosolic receptor for TCDD, and the translooxygenases, together with other enzymes, provide a detoxifi- cation of the inducer-receptor complex to the nucleus is norcation mechanism by which the cell can convert a wide variety mal. The other type variant, which has no detectable basal of of hydrophobic foreign compounds to more polar derivatives, or induced aryl hydrocarbon hydroxylase activity, has a norfacilitating their elimination from the organism (1-3). The mal cytosolic receptor, but is defective in the translocation of activity of many mixed function oxidases is inducible; after the inducer-receptor complex to thenucleus. exposure to an inducer, enzyme activity increases, in some cases 10- to 100-fold. The induction process thus enables the EXPERIMENTAL PROCEDURES AND RESULTS‘ cell to increase its rate of detoxification of lipophilic compounds (4, 5 ) . However, the product(s) of the mixed function DISCUSSION oxidase-catalyzed reaction may be more chemically reactive We have described two classes of mouse hepatoma cell than the parent compound. Therefore, induction of mixed function oxidase activity also may be important in causing variants which contain defective mechanisms of induction of drug toxicity, neoplasia, and developmental malformations (6, the BP-metabolizing enzyme system(s).One class contains an altered cytosolic TCDD receptor which either is decreased in 7). The environmental carcinogen benzo(a)pyrene is a useful amount or is decreased in a f f i t y for the inducer (or both); substrate for analyzing microsomal mixed function oxidase translocation of the inducer-receptor complex from cytoplasm The second induction in cells inculture (8, 9). For example, we have used to nucleus is apparently normal in these variants. the fluorescence-activated cell sorter to studymixed function ’ The abbreviations used are: BP, benzo(a)pyrene; TCDD, 2,3,7,8oxidase activity in intact,viable cells by measuring the rateof
* This research was supported by Research Grants CA 24580 and GM 17367 and Training Grant GM 17149 from the National Institutes of Health and Institutional Grant IN32s from the American Cancer Society. 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. $ Present address, Tumor Virology Laboratory, The Salk Institute, Box 85800,San Diego, CA 92138. 5 Recipient of a Faculty Research Award from the American Cancer Society.
tetrachlorodibenzo-p-dioxin; FACS, fluorescence-activated cell sorter; PEG, polyethylene glycol; PBS, phosphate-buffered saline; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; AHH, aryl hydrocarbon hydroxylase. ‘Portions of this paper (including “Experimental Procedures,” “Results,” Figs. 1-5,and Tables 1-111) 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, cite the authors, and MD 20814. Request Document No. 82M-2288, include a check or money order for $7.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

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class has a cytosolic TCDDreceptor which is normalin tal difference(s)in the induction mechanisms for the two amount andaffinity for inducer; however,translocation of the systems. inducer-receptor complex to the nucleus does not occur in In a preliminary report, Hankinson (31) has described the these variants. Hybrid cells, formed by fusion of cells from the isolation of two classes of BP-resistant variants, which he two classes, contain a normal induction mechanism. Thus, indicated were recessive with respect towild type andwere in these classes represent a t least two complementation groups; different complementation groups. On the basis of the obseradditional intraclass fusions in the future may reveal other vations described here, we have arrived at the same conclucomplementation groups. The phenotype of these variants sion; however, our cell fusion data differ from those of Hanremains stable during months of culture; this is consistent kinson. Our wild type x variant fusions indicate recessive with a mutational origin, as suggested previously by Hankin- inheritance of the variant phenotypes (Table Hankinson’s 11); son (17, 31, 32). are most compatible with co-dominant inheritance (31). In cells The observation that the interclass hybrid contain wild complementation tests, we find complete restoration of wild type levels of aryl hydrocarbon hydroxylase activity presum- type aryl hydrocarbonhydroxylase activity (Table 111); Hanably indicates that at least one the two variant classes has kinson finds 50% or less (31).Therefore, the variants of analyzed an intact structural gene for the enzyme. Since we have no here may differ from those described by Hankinson. reason to think that eitherclass contains multiple defects in Legraverend et al. (25) have recentlyanalyzed the cytosolic its induction mechanism, we assume that the gene coding for TCDD-binding species and its nuclear translocation in several the enzyme is intact in both classes of variant; however, we qf the variants described by Hankinson. They describe one class of variants with defective TCDD binding and normal have not yet demonstrated thisconclusively. In “nonpresonsive” mouse strains, TCDD is able to induce translocation and a second class with normal TCDD binding and defective translocation (25). However, in a variant with hepatic aryl hydrocarbon hydroxylase activity to the same levels observed in “responsive” mice; however, the response decreased cytosolic TCDD binding, Legraverend et al. (25) requires a 10- to 20-fold increase ininducer concentration(33). observed that the EDSo for aryl hydrocarbon hydroxylase induction by TCDD was identical to that in wild type cells. This presumably resultsfrom a cytosolic TCDD receptor which is normal in amount but is decreased in affinity for the This differs from our observations (Fig. 1) and may again inducer (30). In the low activity variants (BP‘c4 and reflect a difference(s) between the variants isolated by HanTAOclBP’cl) described here, TCDDis unable to induce aryl kinson and the variantsdescribed here. The standard approach analyzingexperiments involving for hydrocarbon hydroxylase activity to wild type levels, even at when the inducer is administered a concentration 100-fold cell fusion requires subcloning of the hybrid cells to see if the higher than that required fully induce wild type cells.3This phenotype breeds true and to estimate thedegree of heteroto may mean that the defect in thesecells is primarily a decrease geneity among the hybrid cell population. Such studies are a in their number, rather than in their affinityfor TCDD. laborious and time-consuming; furthermore, during culture, Alternatively, the lack of induction at high TCDD concentra- cell population maybecome heterogeneous with respect to its tions mayreflect the insolubility of TCDD in aqueous media, ability to metabolize BP (11,38,39),complicating the analysis a factor which presumably has less of an effect in whole of results. Use of the FACS minimizes these problems. First, we can analyze cells on the FACS on the same day that we animal experiments. Inthevariantswiththeapparenttranslocation defect assay the bulk population for aryl hydrocarbon hydroxylase of (BP’cl and BP’c3), we do not yet know whether the defect is activity, minimizing artifacts due to the development hetin cytoplasmic ( e g . in an enzyme required for modification of erogeneity during the time culture requiredfor subcloning. Second,theFACS is muchmore efficient. We can easily theinducer-receptor complex prior totranslocation; ina (e.g. “transport” protein required for translocation) or nuclear analyze thousands of cells in minutes on the FACS; it would in a nuclear “acceptor” protein;in a DNA binding sequence). take months to analyze a similar number of subclones using At least one translocation-defective variant (BP’cl) contains the aryl hydrocarbonhydroxylase assay. We believe that the system described here will be useful cell a glucocorticoid-inducible enzyme activity? Since the steroid induction mechanism involves translocation of the inducer- in future studies of the mechanism of induction of the BPreceptor complex from cytoplasm to nucleus (34), this obser- metabolizing enzymesystem. The versatility of the FACS both in the isolation of variants (10) and in the analysis of vation presumably indicates that the translocation lesion in BP’cl is not global, and may be specific to the induction of hybrid cells, as described here, makes an important contribution to our approach. the BP-metabolizing enzyme system. The mechanism of aryl hydrocarbonhydroxylase induction REFERENCES by polycyclic aromatichydrocarbonsis similarin certain 1. Ishimura, Y., Iizuka, T., Morishima, I., and Hayaishi, 0. (1978) in respects to the mechanism of enzyme induction by steroid PolycyclicHydrocarbons and Cancer (Gelboin, H. V., and hormones (7, 34). Other workers havecharacterized cells Ts’o, P. 0. P., eds) pp. 321-322, Academic Press, New York which vary in their response to steroid hormones. They have 2. Sato, R., and Omura, T. (eds) (1978) Cytochrome P-450,Academic isolated variants containing altered steroid receptors and varPress, New York iants with a defect in their translocation mechanism, analo3. Estahrook, R.W., and Werringloer, J. (1979) in The Induction of gous to the two classes which we report here. Furthermore, Drug Metabolism (Estabrook, R.W., and Lindenlaub, E., eds) they havedescribed additional receptor variants which we for pp. 187-199, F. K. Schattauer Verlag, Stuttgart 4. Conney, A, H. (1967) Pharmacol. Rev. 19,317-366 as yet have no counterpart (35,36). However, geneticanalyses ) 178, of the steroid receptor variants using cell fusion indicate that 5. Conney, A. H., and Burns, J. J. (1972) Science (Wash. D. C. 576-586 l they al belong to the same complementation group (36, 37), 6. Gelhoin, H. V., Kinoshita, N., and Wiebel, F. J. (1972) Fed. Proc. a finding which differs from those reported here for the BP31, 1298-1309 resistant variants. We do not know whether this primarily 7. Nehert, D. W., Eisen, H.J., Negishi, M., Lang, M. A., and Hjelmeland, L. M. (1981) Annu. Reu. Pharmacol. Toricol. 21, reflects differences in selection techniques ora fundamenthe

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J A . G. Miller, D. Israel, and J. P. Whitlock, Jr., unpublished observations.

431-462 8. Whitlock, J. P., Jr., and Gelhoin, H. V. (1979) Pharmacol. Ther. 4, 587-599 9. Alfred, L. J., and Gelhoin, H. V. (1967) Science (Wash. D. C.)

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157,75-76 10. Miller, A. G., and Whitlock, J . P., Jr. (1981) J.Biol. Chem. 256, 2433-2437 25. 11. Miller, A. G., and Whitlock, J. P., Jr. (1982) Mol. Cell. Biol. 2, 26. 625-532 12. Miller, A. G., and Whitlock, J . P., Jr. (1982) Cancer Res. 42, 4473-4478 13. Nebert, D. W., and Gelboin, H. V. (1968) J. Biol. Chem. 243, 6242-6249 29. 14. Nebert, D. W., and Gelboin, H. V. (1968) J. Biol. Chem. 243, 6250-6261 30. 15. Poland, A., and Knutson, J . C. (1982) Annu. Pharmacol. Rev. Toxicol. 22,31. 517-554 16. Bernhard, H. P., Darlington, G. J., and Ruddle, F. H. (1973) Deu. B i d . 35,83-96 17. Hankinson, 0. (1979) Proc. Natl. Acad. Sci. U. S. A . 76,373-376 18. Szybalski,W., Szybalska, E. H., and Ragni, G. (1962) Natl. Cancer Monogr. Inst. 7,33. 75-88 19. Littlefield, J. W. (1964) Science (Wash. D. C.) 145, 709-71034. 20. Davidson, R. L., and Gerald, P. S. (1976) SomaticCellGenet. 2, 165-166 21. Corsaro, C. M., and Migeon, B. R. (1978) Somatic Cell Genet. 4, 541-551 22. Okey, A. B., Bondy, G. P., Mason, M. E., Kahl, G. F., Eisen, H. J., Guenthner, T. M., and Nebert, D. W. (1979) J. Biol. Chem. 254, 11636-11648 38. 23. Okey, A. B., Bondy, G. P., Mason, M. E., Nebert, D. W., ForsterGibson, C. J., Muncan, J., and Dufresne, M. J. (1980) J . Biol. Chem. 255, 11415-11422

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24. Hannah, R. R., Nebert, D. W., and Eisen, H. J . (1981) J. Biol. Chem. 256,4584-4590 Legraverend, C., Hannah, R. R., Eisen, H. J., Owens, I. S., Nebert, D. W., and Hankinson, 0. (1982) J . Biol. Chem. 257,6402-6407 Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51,660-672 H., 27. Lowry, 0. Rosebrough, N. J., Farr,A. L., and Randall, R. J. (1951) J.Biol. Chem. 193,265-275 28. Bradford, M. (1976) Anal. Biochem. 72,248-254 Poland, A. P., Glover, E., Robinson, J. Nebert, R., and D. W. (1974) J.Biol. Chem. 249, 5599-5606 Poland, A,, E., Glover, and Kende, A. S. (1976) J.Biol. Chem. 251,4936-4946 Hankinson, 0.(1980) in Microsomes, Drug Oxidations, and Chemical Carcinogenesis (Conney, A. H., Coon, M. J., Estabrook, R. W., Gelboin, H. V., Gillette, J . R., and O’Brien, P. J., e&) pp. 1149-1152, Academic Press, NewYork 32. Hankinson, 0. (1981) Somatic Cell Genet. 7,373-388 Poland, A., and Glover, E. (1975) Mol. Pharmacol. 11,389-398 Baxter, J . D., and Ivarie, R. D. (1978) in Receptorsand Hormone Action (O’Malley, B.W., and Birnbaumer, L., eds) pp. 251-295, Academic Press, New York 35. Sibley, C. H., and Tomkins, G. M. (1974) Cell 2, 221-227 36. Yamamoto, K. R., Gehring, U., Stampfer, M. R., and Sibley, C. H. (1976) Recent Prog. Horm. Res. 32, 3-32 37. Pfahl, M., and Bourgeois, S. (1980) Somatic Cell Genet, 6, 63-74 Whitlock, J . P., Jr., Gelboin, H. V., and Coon, H. G. (1976) J.Cell Biol. 70, 217-225 39. Hankinson, 0. (1980) Somatic Cell Genet. 6,751-767

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