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Binding of Polycyclic Aromatic Hydrocarbons to Transcriptionally Active Nuclear Subfractions of AKR Mouse Embryo Cells¹

Departments of Pathology and Anatomy [H. L. M., G. D. M.] and Molecular Medicine [R. A. W., T. C. S.], Mayo Clinic and Foundation, Rochester, Minnesota 55901

The objective of this study was to examine the binding of carcinogenic polycyclic aromatic hydrocarbons in well-characterized nuclear subfractions from transformable cells in culture. A cloned line of AKR mouse embryo cells was exposed to culture medium containing [³H]-3-methyl-cholanthrene (MC) (0.4 µg/ml) (670 Ci/mole). Cellular uptake and nuclear binding were determined after 4 hr of exposure. The addition of unlabeled MC up to 10 µg/ml did not cause reduction of [³H]MC cellular uptake or nuclear binding. From 2 to 5% of the total cellular MC was localized in the nuclei. All nuclear subfractions obtained from mechanically sheared nuclei and separated on sucrose gradients showed some MC binding; however, a high-affinity, high-specific-activity binding of MC was associated only with the slower-sedimenting component shown to represent that fraction of nuclear chromatin that is transcriptionally active. Conditions that caused the precipitation of this chromatin also resulted in the precipitation of the radioactive compound, thus suggesting that the MC was physically bound to the chromatin. Unlabeled MC (10 µg/ml) saturated this high-affinity MC binding to the transcriptionally active chromatin but did not saturate the binding to the other nuclear fractions. The binding of another potent carcinogen, [³H]-1,2,5,6-dibenzanthracene, and the "weak" carcinogen, [³H]-1,2,3,4-dibenzanthracene (3,4-DBA), to whole nuclei and nuclear subfractions was also determined. The concentration, specific activity, and time of treatment were identical with those used for MC. The level of binding of [³H]-1,2,5,6-dibenzanthracene was approximately 3-fold greater in whole nuclei on a per mass DNA basis than in those of either the MC or the 3,4-DBA. The binding of MC and 3,4-DBA to whole nuclei was approximately equal. As with MC, the [³H]-1,2,5,6-dibenzanthracene demonstrated a peak of high specific activity binding to the slower-sedimenting fraction of chromatin while the 3,4-DBA displayed considerably less binding to this fraction.

¹ This report is Paper 2 of 2 papers on the nuclear subfractions of AKR mouse embryo cells and the binding to them of carcinogenic polycyclic aromatic hydrocarbons. This investigation was supported by Grants CA-16816 and CA-14920 awarded by the National Cancer Institute, Department of Health Education, and Welfare, and the Mayo Foundation.

² To whom requests for reprints should be addressed.

Received 1/15/76. Accepted 4/30/76.