Dehydroepiandrosterone (DHEA) is a 17-ketosteroid that is produced at high levels in the adrenals of primates and is the chief precursor of androstenedione, which itself is readily converted into testosterone and 17β-estradiol at the tissue level [
1]. Epidemiological data have shown that premenopausal women with high levels of DHEA in the serum develop less breast carcinomas than those with low levels of DHEA [
2]. In postmenopausal women DHEA has been hypothesized to contribute to the increased incidence of breast cancer [
3]. Studies on animal model systems revealed that DHEA is a powerful inhibitor of mammary, prostate, skin, lung, liver, and thyroid carcinogenesis [
4‐
7]. DHEA and its fluorinated analogue, DHEA 8354, given after carcinogen administration, inhibited mammary carcinogenesis in rats in a dose-dependent manner [
6,
7]. DHEA combined with 4-(Hydroxyphenyl)retinamide (4-HPR) further decreased the incidence and frequency of mammary tumors in an
N-nitroso-
N-methylurea (MNU) carcinogenesis model [
4]. Tamoxifen (0.08 mg/kg diet) combined with low-dose DHEA (400 mg/kg diet) further decreased the incidence and multiplicity of mammary tumors [
6]. DHEA also decreased the incidence of mammary tumors in rats injected with dimethylbenz(a)anthracene (DMBA) into mammary gland parenchyma and continuously stimulated with prolactin [
8]. Studies with C3(1)/SV40 Tag mice, which can spontaneously develop estrogen receptor (ER)-negative mammary and prostate tumors, have shown that DHEA at 4,000 mg/kg diet given between the 7 th and 19 th weeks of age, decreased the incidence of mammary tumors by 30% and tumor multiplicity by about 50% [
9]. Furthermore, in both DHEA-treated C3(1)/SV40 Tag mice and DHEA-treated rats the circulating estradiol levels were increased, suggesting the involvement of a tumor suppressor mechanism that is not directly related to the ER signaling. DHEA was also effective in inhibiting the growth of ZR-75-1 ER-positive breast cancer cells transplanted into nude mice [
10]. In most of these studies, DHEA was given in the diet 1 week before and/or continuously after carcinogen administration, until the animals were killed [
4‐
6]. However, with this approach the limited number of tumors that occur in DHEA-treated animals are apparently resistant to DHEA and therefore the biomarkers that would be identified might not reflect the preventive and antitumor potential of DHEA in the course of tumor development. Further, the molecular mechanism of action of DHEA remains poorly defined.
Here we have examined the effect of DHEA and its analog DHEA 8354, initiated 6 weeks after carcinogen administration, when hyperplastic and premalignant lesions occur in the mammary gland, on the following: first, the incidence, multiplicity and weight (burden) of mammary tumors; second, the proliferative activity of tumor cells; third, apoptosis; fourth, cellular senescence; fifth, cell cycle distribution; and sixth, the expression of p53, p21, and p16. To identify senescent cells in mammary tissues and tumors, a panel of methods was employed, including senescence-associated β-galactosidase (SA-β-Gal) staining [
11], continuous labeling with bromodeoxyuridine (BrdU) [
12], analysis of 90° light scatter by flow cytometry, and cytomorphological criteria [
13]. Because p53, p21, and/or p16 genes are key mediators for the initiation and maintenance of cellular senescence and apoptosis [
14‐
17], their role in such mechanisms was investigated. It has been recently observed that p16
INK4A is overexpressed in senescent cells, suggesting that it might be upregulated in mammary tumors treated with DHEA [
18]. We provide comprehensive evidence of a correlation between the inhibitory effects of DHEA on mammary carcinogenesis, induction of cellular senescence, inhibition of cell proliferation, induction of apoptosis and the expression of p21 and p16
INK4A.