Growth factors and growth inhibitors play crucial roles in the growth regulation and differentiation of mammary epithelial cells. Studies have shown that during pregnancy, with the onset of terminal differentiation, there is a dramatic decrease in the proliferation of the mammary epithelial cells. Here we report the cloning and characterization of a novel pregnancy-induced cDNA, OKL38, from a human ovarian cDNA library. This cDNA encodes for a protein of approximately 34.5 kDa. Tissue distribution studies through Northern analyses revealed the ubiquitous nature of OKL38 transcripts in most tissues, with the highest levels observed in the ovary, kidney, and liver. The onset and advancement of pregnancy also gave rise to a concomitant increase in OKL38 gene expression. In situ hybridization revealed that OKL38 mRNA was further detected in mammary secretory epithelial cells. However, low levels of OKL38 transcripts were observed in the various human breast cancer cell lines studied and were barely detectable in all dimethylbenz(A)anthracene-induced mammary tumors examined. Transfection studies with OKL38 cDNA with MCF-7 cells resulted in growth inhibition in vitro and reduction in tumor formation in vivo. These observations led to speculation that OKL38 may play a vital role in the growth regulation and differentiation of breast epithelial cells during pregnancy and its implications in tumorigenesis.

Abstract. Many environmental chemicals including pesticides have been reported to possess hormonal activities, and thus are classified as endocrine disruptors. Permethrin, a synthetic pyrethroid insecticide, is used worldwide, which provides potential environmental exposure. However, relatively few studies have reported on hormonal activities, particularly estrogenic and androgenic activities of permethrin, and the results of these studies are in some respects contradictory. Therefore, this study investigated the potential estrogenic and androgenic activities of permethrin in vitro and in vivo. We conducted an uterine Calbindin-D9k (CaBP-9k) gene expression assay and an uterotrophic assay for estrogenic activity, and a Hershberger assay for androgenic activity. The CaBP9k gene, one of the intracellular calcium binding proteins, is estrogen-responsive in the uterus. The rat uterotrophic and Hershberger assays are generally used as in vivo short-term screening assays for detecting the estrogenic and androgenic activities of chemicals, although these assays are still being validated by the Organization for Economic Cooperation and Development (OECD). Northern blot analysis showed the induction of uterine CaBP-9k mRNA level in response to permethrin as well as co-administration of permethrin with E2. In the uterotrophic assay using 18-day-old female rats, subcutaneous treatments with permethrin (10 to 800 mg/kg) for three days increased relative uterine wet weights, and E2-induced uterine weights. These effects were statistically significant at 800 and 200 mg/kg, respectively. Moreover, permethrin-induced uterine weights were inhibited by the coadministration of ICI 182,780, an antiestrogen. In the Hershberger assay, the administration of permethrin orally to testosterone propionate-treated castrated male rats led to statistically significant reductions in androgen-dependent sex accessory tissue (ventral prostate, seminal vesicles, levator ani and bulbocavernosus muscles, Cowper's gland and glans penis) weights at all doses tested (10, 50 and 100 mg/kg). These results suggest that permethrin might have estrogen-like effects on female rats, but antiandrogen-like effects on males.

The antiestrogen, ICI 182780 (ICI) proves to be clinically useful for the treatment of estrogen receptor positive breast tumours. We report the assessment of the in vivo and in vitro effects of ICI on apoptosis of breast epithelial cells. In vivo, administration of rats with ICI for 3 weeks resulted in a reduction in the size of the lobular structures with the rate of mammary epithelial apoptosis equivalent to 10, 35 and 45% on treatment with 1, 1.5 and 2 mg ICI per kg body weight, respectively. Concomitantly, these treatment led to a 2.0-, 2.2- and 2.5-fold increase in Bax. Similar elevations were also observed in Bad levels which increased 1.7-, 2.6- and 2.7-fold respectively in the ICI treatment as compared to controls. This also resulted in a dose dependent decrease in Bcl-2 and Bcl-xL protein expressions. Growth inhibition and induction of apoptosis were also observed in the MCF-7 cells following in vitro treatment with ICI. This is closely associated with [1] the down-regulation of Bcl-2 and Bcl-xL proteins and [2] upregulation of Bax and Bad, whose gene products are known to be involved the regulation of apoptosis in mammalian cells. Stable over-expression of Bcl-2 resulted in protection of MCF-7 cells from apoptosis and growth inhibitory effects of ICI. Conversely, reduction of Bcl-2 by antisense transfection make MCF-7 cells more sensitive to ICI-induced growth inhibition and apoptosis. These findings suggest that modulation of Bax, Bcl-xL, Bcl-2 and Bad proteins by ICI may be, in part, responsible for the anti-proliferative and apoptotic effect of ICI seen clinically and in animal models.

A complementary DNA, uterine-ovarian-specific gene 44 (UO-44), has been isolated from tamoxifen-induced rat uterine complementary DNA library using differential display techniques. UO-44 transcripts are found to be abundant in the uterus and ovary. UO-44 gene expression in the uterus is strictly regulated by estrogens, tamoxifen, and GH, whereas the pure antiestrogen ICI 182780 is inhibitory. Treatment of ovariectomized rats and hypophysectomized rats with tamoxifen and GH, respectively, resulted in up-regulation of UO-44 expression in a dose-dependent manner. In situ hybridization revealed that UO-44 gene expression was restricted to the luminal and glandular epithelial cells of the uterus and to granulosa cells of medium-size ovarian follicles. Transfection studies showed that UO-44 was a membrane-associated protein. Because estrogens, tamoxifen, and GH are stimulators of uterine luminal epithelial cell growth in vivo, UO-44 protein may serve as a mediator of the effect of these compounds in inducing epithelial proliferation and differentiation in these tissues.