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Reversal of Cytochrome P-4501A1 and P-450-EF Expression in MCA-C3H/10T1/2 Cell-derived Tumors as Compared to Cultured Cells¹

Department of Pharmacology, Medical Sciences Center [M. C., X. S., C. R. J.], and Department of Comparative Biosciences, School of Veterinary Medicine [I. M. K., M. E. S.], University of Wisconsin, Madison, Wisconsin 53706

The 3-methylcholanthrene-transformed tumorigenic cell line, MCA-C3H/10T1/2 CL15 (MCA), expresses the novel benz(a)anthracene (BA)-inducible polycyclic aromatic hydrocarbon-metabolizing cytochrome P-450 (P-450-EF). The level of expression is comparable to that reported for the nontumorigenic C3H/10T1/2 CL8 (10T1/2) cells (Pottenger, L. H., Christou, M., and Jefcoate, C. R. Arch. Biochem. Biophys., 286: 488–497, 1991). Sarcomas (3–12 mm in diameter) generated in athymic "nude" mice by s.c. injection of MCA cells exhibited much lower 7,12-dimethylbenz-(a)anthracene-metabolizing activites (5–15% of the levels in cultured cells), both constitutively and after in vivo treatment with BA. A sharp decrease in P-450-EF expression was observed both at the functional level (10- to 30-fold) (as determined by antibody inhibition studies) and at the apoprotein level (50- to > 100-fold) (as determined by Western immunoblots). However, in contrast to the BA-treated MCA cells in which P-450-EF comprises essentially the total spectrally detectable P-450 content (30 pmol/mg) with virtually undetectable cytochrome P-4501A1, tumors from these cells expressed substantial levels of P-4501A1 immunodetectable protein in response to BA treatment (0.5–3 pmol/mg). In these tumors, P-450-EF expression decreased to undetectable or barely detectble levels (<0.2–0.5 pmol/mg), P-4501A1-expressing cells were localized in tumor sections immunocytochemically and were morphologically identical to other MCA cells, which formed the majority of the sarcoma. At the functional level, antibody inhibition studies and product ratios demonstrated that P-4501A1 accounted for only 40% of the total dimethylbenz(a)anthracene-metabolizing activity of BA-induced tumor microsomes, whereas the remaining activity was due to P-450-EF. This low catalytic activity for P-4501A1 (35–95 pmol/mg/h) indicated that the majority of BA-inducible P-4501A1 in the tumors (>95%) was expressed as apoprotein. The contribution from P-450-EF was consistent with full expression of hemoprotein. Tumor size affected the total dimethylbenz(a)-anthracene-metabolizing activities/mg microsomal protein (small tumors were 2- to 3-fold more active than large tumors) but had no effect on the ratio of activites dependent on, respectively, P-450-EF and P-4501A1 holoenzymes (1.5:1), thus suggesting controlled coexpression of these proteins. Reculturing of tumor-derived cells effected partial restoration of P-450-EF expression and eliminated the expression of P-4501A1, confirming a unique contribution from tumor environment to the regulation of these genes. Possible mechanisms for an environment-dependent concerted regulation of the P-4501A1 and P-450-EF genes are discussed.

¹ This research was supported by National Institutes of Health Grant CA16265 and in part by the Council for Tobacco Research U.S.A., Inc., Grant 2963.

² To whom requests for reprints should be addressed, at the Department of Pharmacology, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706.

Received 7/ 8/92. Accepted 12/21/92.

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