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Experimental down-regulation of intermediate biomarkers of carcinogenesis in mouse mammary epithelial cells

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Summary

The polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) is a metabolism-dependent procarcinogen whose tumorigenicity is modified by dietary and endocrine manipulationsin 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 (E2) metabolites onin vitro DMBA carcinogenesis utilizing a newly established mouse mammary epithelial cell line C57/MG. Alteration in DNA repair synthesis, metabolism of E2 via the C2- and C16α-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 E2 metabolites. A single 24 hr exposure of 0.78 µM DMBA to C57/MG cells resulted in a 193.9% increase in DNA repair synthesis and a 73.1% decrease in C2/C16α hydroxylation of E2. The DMBA treated C57/MG cells also exhibited increased anchorage-independencein vitro prior to tumorigenesisin vivo. A simultaneous treatment of cells with DMBA and with the highest non-cytotoxic doses of the tumor suppressing agents 5 µM N-(4-hydroxyphenyl) retinamide (HPR), 50 µM indole-3-carbinol (I3C), or 1 µM tamoxifen (TAM) resulted in a 35.6% to 63.9% decrease in DNA repair synthesis, a 23.8% to 1347.6% increase in C2/C16α hydroxylation of E2, and a 53.8% to 72.4% decrease in anchorage-independent growth. The E2 metabolites at the highest non-cytotoxic doses of 0.76 µM estrone (E1), 0.69 µM 2-hydroxyestrone (2-OHE1), and 0.66 µM 2-methoxyestrone (2-MeOHE1) suppressed DMBA-induced DNA repair synthesis by 56.0% to 68.8%. These tumor suppressing agents and E2 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/C16α hydroxylation in favor of C16α-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 E2 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.

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Abbreviations

DMBA:

7,12-dimethylbenz(a)anthracene

HPR:

N-(4-hydroxyphenyl) retinamide

I3C:

indole-3-carbinol

TAM:

tamoxifen

E2 :

17β-estradiol

E1 :

estrone

2-OHE1 :

2-hydroxyestrone

2-MeOHE1 :

2-methoxyestrone

16α-OHE1 :

16α-hydroxyestrone

E3 :

estriol

DME/F12:

Dulbecco's modified Eagle's medium

F12:

Ham's medium

HU:

hydroxyurea

PBS:

phosphate buffered saline

NaOH:

sodium hydroxide

SDS:

sodium dodecyl sulfate

TCA:

trichloroacetic acid

[C2-3H] E2 :

estradiol labeled at C2 position

[C16α-3H] E2 :

estradiol labeled at C16α position

ANOVA:

analysis of variance

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Authors and Affiliations

  1. Strang-Cornell Cancer Research Laboratory, Cornell University Medical College, New York, USA

    A. Suto, H. Leon Bradlow, Michael P. Osborne & Nitin T. Telang

  2. Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, USA

    George Y. C. Wong

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  1. A. Suto
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  2. H. Leon Bradlow
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  3. George Y. C. Wong
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  4. Michael P. Osborne
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  5. Nitin T. Telang
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Suto, A., Bradlow, H.L., Wong, G.Y.C. et al. Experimental down-regulation of intermediate biomarkers of carcinogenesis in mouse mammary epithelial cells. Breast Cancer Res Tr 27, 193–202 (1993). https://doi.org/10.1007/BF00665689

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