EXPERIMENTAL DOWN-REGULATION OF INTERMEDIATE BIOMARKERS OF CARCINOGENESIS IN MOUSE MAMMARY EPITHELIAL-CELLS

The polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) is a metabolism-dependent procarcinogen whose tumorigenicity is modified by dietary and endocrine manipulations in vivo. DMBA initiates molecular and cellular alterations in the mammary tissue, while dietary components and estrogens affect the post-initiational phase of tumorigenic transformation. The mechanism(s) responsible for modulation of tumorigenic transformation remain unclear. This study examines the effects of selected tumor suppressing agents and estradiol (E(2)) metabolites on in vitro DMBA carcinogenesis utilizing a newly established mouse mammary epithelial cell line C57/MG. Alteration in DNA repair synthesis, metabolism of E(2) via the C2- and C16 alpha-hydroxylation pathways, and acquisition of anchorage-independent growth were utilized as molecular, endocrine, and cellular biomarkers to quantitate the cellular transformation by DMBA and its modulation by tumor suppressing agents and E(2) metabolites. A single 24 hr exposure of 0.78 mu M DMBA to C57/MG cells resulted in a 193.9% increase in DNA repair synthesis and a 73.1% decrease in C2/C16 alpha hydroxylation of E(2). The DMBA treated C57/MG cells also exhibited increased anchorage-independence in vitro prior to tumorigenesis in vivo. A simultaneous treatment of cells with DMBA and with the highest noncytotoxic doses of the tumor suppressing agents 5 mu M N-(4-hydroxyphenyl) retinamide (HPR), 50 mu M indole-3-carbinol (I3C), or 1 mu M tamoxifen (TAM) resulted in a 35.6% to 63.9% decrease in DNA repair synthesis, a 23.8 % to 1347.6% increase in C2/C16 alpha hydroxylation of E(2), and a 53.8% to 72.4% decrease in anchorage-independent growth. The E(2) metabolites at the highest non-cytotoxic doses of 0.76 mu M estrone (E(1)), 0.69 mu M 2-hydroxyestrone (2-OHE(1)), and 0.66 mu M 2-methoxyestrone (2-MeOHE(1)) suppressed DMBA-induced DNA repair synthesis by 56.0% to 68.8%. These tumor suppressing agents and E(2) metabolites also effectively suppressed post-initiational, anchorage-independent growth by 24.9% to 72.4%. These results indicate that DMBA induces cellular transformation in part by causing DNA damage, altering C2/C16 alpha hydroxylation in favor of C16 alpha-hydroxylation, and inducing anchorage-independent growth prior to tumor development. Effective downregulation of these genotoxic, endocrine and proliferative end points by prototypic tumor suppressing agents and by E(2) metabolites generated via the C2-hydroxylation pathway suggest that these agents may influence mammary tumorigenesis by inhibiting early occurring initiational and/or post initiational events.