Abstract
Purpose. To develop Caco-2 cell derivatives expressing high levels of human cytochrome P450 drug metabolizing enzymes.
Methods. The cDNAs for two cytochrome P450 forms, CYP2A6 and CYP3A4, were introduced into an extrachromosomal vector under control of the cytomegalovirus early intermediate promoter. Vector-bearing cells were selected via resistance to hygromycin B.
Results. Transfected cells exhibited high levels of cDNA-derived protein as measured by Western blot, spectrophotometric P450 determination and/or cytochrome P450 form-selective enzyme assay. CYP3A4 and CYP2A6 catalytic activities were about 100 fold higher than in control cells. cDNA-expressing cells were found to form tight monolayers and were suitable for study of xenobiotic transport and metabolism. The permeabilities of cephalexin, phenylalanine, mannitol and propranolol across transfected monolayers were found to be similar to those across untransfected monolayers. The appropriate transfected monolayers metabolized the CYP2A6 substrate coumarin and the CYP3A4 substrates testosterone and nifedipine.
Conclusions. A Caco-2 cell system to simultaneously study drug transport and metabolism has been developed.
Similar content being viewed by others
REFERENCES
Pinto, M., Robine-Leon, S., Appay, M. D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assman, P., Haffen, K., Fogh, J., and Zweibaum, A. Biol. Cell 47:323–330 (1983).
Hidalgo, I. J., Raub, T. J., and Borchardt, R. T. (1989). Gastroenterology, 96:736–749.
Wilson, B., Hassan, I. F., Dix, C. J., Williamson, I., Shah, R., Mackay, M., and Artursson, P. J. Control. Rel. 11:25–40 (1990).
Artursson, P. and Karlsson, J. Biochem. Biophys. Res. Comm. 175:880–885 (1991).
First, M. R., Schroeder, T. J., Alexander, J. W., Stephens, G. W., Weiskittel, P., Myre, S. A., and Pesci, A. J. Transplantation, 51:365–370 (1991).
Yun, C. H., Okerholm, R. A., and Guengerich, F. P. Drug Metab. Disp. 21:403–409 (1993).
Watkins, P. B., Wrighton, S. A., Scheutz, E. G., Molowa, D. T. and Guzelian, P. S. J. Clin. Invest. 80:1029–1036 (1987).
Kolars, J. C., Awni, W. M., Merionand, R. M., and Watkins, P. B. Lancet, 338:1488–1490 (1991).
Kolars, J. C., Schmiedlin-Ren, P., Scheutz, J. D., Fang, C., and Watkins, P. B. J. Clin. Invest. 90:1871–1878 (1992).
Yates, J. L., Warren, N., and Sugden, B. Nature (Lond.), 313:812–815 (1985).
Crespi, C. L., Penman, B. W., Leakey, J. A. E., Arlotto, M. P., Stark, A., Turner, T., Steimel, D., Rudo, K., Davies, R. L., and Langenbach, R. L. Carcinogenesis, 8:1293–1300 (1990).
Crespi, C. L., Penman, B. W., Steimel, D. T., Gelboin, H. V., and Gonzalez, F. J. Carcinogenesis, 12:355–359 (1991).
Penman, B. W., Reece, J., Smith, T., Yang, C. S., Gelboin, H. V., Gonzalez, F. J., and Crespi, C. L. Pharmacogenetics, 3:28–39 (1993).
Omura, T. and Sato, R. J. Biol. Chem. 239:2379–2385 (1964).
Phillips, A. H. and Langdon, R. G. J. Biol. Chem. 237:2652–2660 (1962).
Greenlee, W. F. and Poland, A. J. Pharmacol. Exp. Ther. 205:596–605 (1978).
Yamano, S., Tatsumo, J., and Gonzalez, F. J. Biochem. 29:1322–1329 (1990).
Waxman, D. J., Ko, A., and Walsh, C. J. Biol. Chem. 258:11937–11947 (1983).
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, F. J. J. Biol. Chem. 62:315–323 (1951).
Hu, M., Chen, J., Zhu, Y., Dantzig, A. H., Stratford, R. E., and Kuhfeld, M. T. Pharm. Res. 11:1405–1413 (1994).
Hu, M., Chen, J., Tran, D., Zhu, Y., and Leonardo, G. J. Drug Targeting, 2:79–89 (1994).
Hu, M., Zheng, L., Liu, L., Dantzig, A. H., and Stratford Jr., R. E. J. Drug Targeting, 3:291–300 (1995).
Bradford, M. (Biochem. Pharmacol. 47:1957–1963 1976).
Crespi, C. L., Steimel, D. T., Aoyama, T., Gelboin, H. V., and Gonzalez, F. J. Molec. Carcinogenesis, 3:5–8 (1990).
Sugden, B., Marsh, K., and Yates, J. Molec. Cell Biol. 5:410–413 (1985).
Pearce, R., Greenway, D., and Parkinson, A. Arch. BioChem. Biophys. 298:211–225 (1992).
Penman, B. W., Chen, L. P., Gelboin, H. V., Gonzalez, F. J., and Crespi, C. L. Carcinogenesis, 15:1931–1937 (1994).
Wrighton, S. A., Van den Branden, M., Stevens, J. C., Shipley, L. A., and Ring, B. J. Drug Metab. Rev. 25:453–484 (1993).
Carriere, V., Lesuffleur, T., Barbat, A., Rousset, M., Dussaulx, E., Costet, P., Dewaziers, I., Beaune, P., and Zweibaum, A. FEBS Letters, 355:247–250 (1994).
Gan, L.-S., Moseley, A., Khosla, B., Augustijins, P., Bradshaw, T., Hendren, W., and Thakker, D. Drug Metab. Disp. 24:344–349 (1996).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Crespi, C.L., Penman, B.W. & Hu, M. Development of Caco-2 Cells Expressing High Levels of cDNA-Derived Cytochrome P4503A4. Pharm Res 13, 1635–1641 (1996). https://doi.org/10.1023/A:1016428304366
Issue Date:
DOI: https://doi.org/10.1023/A:1016428304366