Identification of human estrogen-inducible transcripts that potentially mediate the apoptotic response in breast cancer.

Hormone manipulation has been used for several decades with the purpose of inducing breast cancer regression. On the one hand, hormone ablation and antiestrogen administration were used on the rationale that estrogens induce proliferation of their target cells. Before the advent of the antiestrogen tamoxifen, on the other hand, the estrogen agonist DES was used to obtain clinical remissions. The rationale for the use of diethylstilbestrol (DES) was totally empirical. In fact, the efficacy of both treatments was comparable. A mechanistic explanation for estrogen-induced regression is urgently needed in order to provide a rationale for its use in therapeutic fields, and to develop markers to identify this phenotype in order to recognize responsive tumors. In this report, we use E8CASS cells (a MCF7 variant) as a model to study estrogen-mediated regression. The proliferation rate of E8CASS cells is decreased by estrogens. In order to isolate mRNA sequences induced by estradiol, a subtracted library was prepared from E8CASS cells grown in the presence and absence of estrogens. Twenty nine differentially expressed unique sequences were found. Seven of them were homologous to known genes, 12 of them were homologous to expressed sequence tags (EST), and 10 sequences had no homologues in the databases. The two sequences showing the highest induction by estradiol (E9 and E43) were chosen for further analysis. The sequence of the E43 coding region has 96% homology to the bovine actin2 gene and 100% identity to bovine actin2 protein, and it is homologous to the human actin-related protein 3 (Arp3). It has been suggested that Arp3 is involved in actin nucleation. The phenotype of E8CASS cells is clearly affected by estrogen treatment. It is likely that E43 may be involved in these morphological changes. The E9 cDNA is a putative zinc-finger protein of the PHD family of transcriptional transactivators. A member of this family, Requiem, is involved in apoptosis. The E9 mRNA is highly expressed in E8CASS cells treated with estrogens, a treatment which results in decreased proliferation rate and increased DNA degradation. This correlation suggests that E9 may be a mediator of estrogen-induced regression of breast cancer.

Assays to measure estrogen and androgen agonists and antagonists.

Substantial evidence has surfaced on the hormone-like effects of many xenobiotics in fish, wildlife and humans (Colborn et al., 1993). The endocrine and reproductive effects of xenobiotics are believed to be due to their 1) mimicking the effects of endogenous hormones such as estrogens and androgens, 2) antagonizing the effects of normal, endogenous hormones, 3) altering the pattern of synthesis and metabolism of natural hormones, and 4) modifying the hormone receptor levels. Estrogen mimics (xenoestrogens) are among the environmental chemicals found to cause reproductive impairment in wildlife and humans. All of these compounds were found to be estrogenic long after they had been released into the environment. A single causal agent can be identified in cases in which humans have had occupational exposures, whereas in cases where wildlife have shown signs of reproductive damage the exposure is usually a combination of endocrine disruptors that may have acted cumulatively. It has been hypothesized that environmental estrogens may play a role in the decrease of human semen quantity and quality of human semen during the last 50 years. They may also be partly responsible for the increased incidence of testicular cancer and cryptorchidism in males and breast cancer incidence in both females and males in the industrialized word. Testing this hypothesis will require: 1) the identification of xenoestrogens among the chemicals present in the environment, 2) the development of a methodology to assess the interactions among mixtures of xenoestrogens to which humans are exposed, and 3) the discovery of markers of estrogen exposure. The development of fast and sensitive bioassays is central to the achievement of these three goals.