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Expression of Pregnancy-specific Genes in Preneoplastic Mouse Mammary Tissues from Virgin Mice¹

Laboratory of Molecular Biology [G. H. S., D. E. G.], and Laboratory of Pathophysiology [B. K. V.], National Cancer Institute, Bethesda, Maryland 20205; and Department of Cell Biology [D. M.], Baylor College of Medicine, Houston, Texas 77030

Experimentally induced breast cancer is often preceded by the appearance of preneoplastic lesions which possess the attributes of hyperplastic normal tissue. These lesions can be isolated and carried as stably transplantable outgrowth lines which continue to morphologically resemble differentiating mammary tissue (Medina, D. Methods Cancer Res., 7: 3–53, 1973). We established seven serially transplantable hyperplastic alveolar nodule (HAN) outgrowth lines from virgin mouse mammary tissues following induction by mouse mammary tumor virus, dimethylbenz(a)-anthracene, and/or pituitary isografts. The expression of mammary differentiation-specific casein genes was measured in these hyperplastic outgrowths by immunocytochemistry, specific radioimmune precipitation, and blot hybridization of total RNA. All seven HAN outgrowth lines were immunologically positive for casein both in situ and upon explant culture. Unlike explants from normal virgin mouse mammary gland, exposure to insulin, hydrocortisone, and prolactin induced an increase in casein synthesis in HAN explant cultures which was independent of DNA synthesis. [³⁵S]Methionine-labeled polypeptides synthesized in explant cultures of HAN outgrowths freshly isolated from virgin hosts were analyzed by radioimmune precipitation and gel electrophoresis. This analysis demonstrated that all major species of casein, (M_r 46,000), ß (M_r 27,000), and (M_r 25,000), were constitutively (i.e., in the absence of lactogenic stimuli) expressed in these preneoplastic alveolar mammary outgrowths. In support of this observation, RNA homologous to ß- and - casein cDNA probes was often detectable in total RNA preparations from freshly isolated fragments of HAN outgrowths.

A second mammary differentiation specific gene product, -lactalbumin, was also detected in HAN outgrowths both in situ and following explant culture. Enzymatically active -lactalbumin was present in extracts of freshly isolated HAN outgrowth tissues and was detectable in these same tissues by immunoperoxidase. In general, -lactalbumin synthesis was increased during explant culture in the presence of lactogenic hormones; however, in contrast to casein synthesis, insulin-hydrocortisoneprolactin-induced increase in -lactalbumin production in vitro was occasionally dependent upon DNA synthesis as it is in explants from normal virgin mouse mammary tissue.

The significant accumulation of casein and -lactalbumin in these hyperplastic virgin mouse mammary cells in nonpregnant hosts suggests that at least two hormonally controlled differentiation-specific genes are constitutively expressed as a result of preneoplastic transformation. Of additional interest was the observation that the levels of casein and -lactalbumin were elevated in the normal mammary tissues of the virgin hosts carrying the preneoplastic mammary outgrowths. Whether this activity is a manifestation of hormonal imbalances resulting from the transplantation of preneoplastic tissue into the cleared mammary fat pad or specifically induced by cellular products of the transformed mammary epithelium is at present unclear.

¹ This research was partially supported by Contract No-1-CP 9-1020 from the National Cancer Institute.

² To whom requests for reprints should be addressed.

Received 12/27/83. Accepted 4/13/84.

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