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Isolation and Characterization of Polyploid Nuclei of R3230AC Mammary Adenocarcinoma and Comparison with Normal Mammary Nuclei¹

Department of Pharmacology and Specialized Cancer Research Center, The Milton S. Hershey Medical Center, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

The R3230AC mammary adenocarcinoma is biochemically similar to the normal lactating mammary gland in many ways, including the RNA/DNA ratio. However, the tumor differs from normal in important ways, which include having a DNA content 2 to 4 times higher than the maximum value for normal mammary gland. This raises the question of whether the cells of the tumor all have more DNA than normal cells. In order to determine how tumor DNA is distributed and whether its transcriptional activity differs from normal, we have compared the ploidy and RNA polymerase activities of nuclei isolated from both sources. Nuclei were also examined for their content of Ca²⁺-stimulated RNase activity, a marker of mammary differentiation.

Sucrose density gradient centrifugation of tumor cell nuclei revealed three nuclear types, designated T1, T2, and T3, representing 25, 48, and 27% of total nuclei isolated. T1, T2, and T3 nuclei contained, respectively, 6.5, 12.3, and 19.0 pg DNA per nucleus, representing diploid, tetraploid, and hexaploid amounts of DNA. Nuclear RNA and protein increased in proportion to ploidy. By contrast, cell nuclei isolated from lactating mammary gland formed only two bands (M1 and M2), comprising 54 and 46%, respectively, of total nuclei isolated. Both M1 and M2 nuclei contained diploid amounts of DNA. Therefore, the higher DNA content in the tumor is attributable, at least in part, to the presence of polyploid nuclei in the majority of tumor cells.

The combined activity of DNA-dependent RNA polymerases I, II, and III per nucleus was the same in T1, T2, and T3; M1 and M2 also had activities similar to each other but much lower than the tumor types. Activity attributable to polymerase I (resistant to 200 µg -amanitin per ml), polymerase II (inhibited by 0.06 µg -amanitin per ml), and polymerase III (inhibited by 200 µg -amanitin per ml) was about 35, 50, and 15%, respectively, for each class of tumor nuclei. The corresponding values for M bands were about 44, 34, and 22%. These results suggest that tumor cells synthesize more heterogeneous RNA than do mammary cells of lactating rats, but the latter synthesize more ribosomal RNA. Activity of mammary-specific Ca²⁺-stimulated RNase increased slightly per nucleus as the ploidy of tumor nuclei increased, but in every T group the activity was less than one-half of that of M1 or M2 nuclei. Thus, despite differences in ploidy, all tumor nuclear classes show a lesser degree of differentiation than nuclei from the lactating gland, with respect to their capacity for synthesis of ribosomal RNA compared to messenger RNA and their expression of Ca²⁺-stimulated RNase.

¹ Supported in part by USPHS Grants CA18200 and AM17644 from the NIH and Grant IN-109 from the American Cancer Society.

² To whom requests for reprints should be addressed.

Received 1/12/81. Accepted 7/10/81.