Genistein effects on stromal cells determines epithelial proliferation in endometrial co-cultures.

BACKGROUND: Estrogen is the leading etiologic factor for endometrial cancer. Estrogen-induced proliferation of endometrial epithelial cells normally requires paracrine growth factors produced by stromal cells. Epidemiologic evidence indicates that dietary soy prevents endometrial cancer, and implicates the phytoestrogen genistein in this effect. However, results from previous studies are conflicting regarding the effects of genistein on hormone responsive cancers. METHODS: The effects of estrogen and genistein on proliferation of Ishikawa (IK) endometrial adenocarcinoma cells were examined in co-cultures of IK cells with endometrial stromal cells, recapitulating the heterotypic cell-to-cell interactions observed in vivo. The roles of estrogen receptor (ER)α and ERß were evaluated using ERα and ERß specific agonists. ER activation and cell proliferation in the IK epithelial cells were determined by alkaline phosphatase assay and Coulter counter enumeration, respectively. RESULTS: Both estrogen and genistein increased estrogen receptor-induced gene activity in IK cells over a range of concentrations. Estrogen alone but not genistein increased IK proliferation in co-cultures. When primed by estrogen treatment, increasing concentrations of genistein produced a biphasic effect on IK proliferation: nM concentrations inhibited estrogen-induced proliferation while µM concentrations increased proliferation. Studies with an ERß-specific agonist produced similar results. Genistein did not influence the effects of estrogen on IK proliferation in monoculture. CONCLUSIONS: Our study indicates that nutritionally relevant concentrations (nM) of genistein inhibit the proliferative effects of estrogen on endometrial adenocarcinoma cells presumably through activation of stromal cell ERß. We believe that sub-micromolar concentrations of genistein may represent a novel adjuvant for endometrial cancer treatment and prevention.

Gene expression patterns associated with p53 status in breast cancer.

BACKGROUND: Breast cancer subtypes identified in genomic studies have different underlying genetic defects. Mutations in the tumor suppressor p53 occur more frequently in estrogen receptor (ER) negative, basal-like and HER2-amplified tumors than in luminal, ER positive tumors. Thus, because p53 mutation status is tightly linked to other characteristics of prognostic importance, it is difficult to identify p53's independent prognostic effects. The relation between p53 status and subtype can be better studied by combining data from primary tumors with data from isogenic cell line pairs (with and without p53 function). METHODS: The p53-dependent gene expression signatures of four cell lines (MCF-7, ZR-75-1, and two immortalized human mammary epithelial cell lines) were identified by comparing p53-RNAi transduced cell lines to their parent cell lines. Cell lines were treated with vehicle only or doxorubicin to identify p53 responses in both non-induced and induced states. The cell line signatures were compared with p53-mutation associated genes in breast tumors. RESULTS: Each cell line displayed distinct patterns of p53-dependent gene expression, but cell type specific (basal vs. luminal) commonalities were evident. Further, a common gene expression signature associated with p53 loss across all four cell lines was identified. This signature showed overlap with the signature of p53 loss/mutation status in primary breast tumors. Moreover, the common cell-line tumor signature excluded genes that were breast cancer subtype-associated, but not downstream of p53. To validate the biological relevance of the common signature, we demonstrated that this gene set predicted relapse-free, disease-specific, and overall survival in independent test data. CONCLUSION: In the presence of breast cancer heterogeneity, experimental and biologically-based methods for assessing gene expression in relation to p53 status provide prognostic and biologically-relevant gene lists. Our biologically-based refinements excluded genes that were associated with subtype but not downstream of p53 signaling, and identified a signature for p53 loss that is shared across breast cancer subtypes.

Expression of exogenous human telomerase in cultures of endometrial stromal cells does not alter their hormone responsiveness.

In the human endometrium, stromal cells mediate the proliferative response of epithelial cells to the steroid hormones estrogen and progesterone. These stromal-epithelial interactions are readily studied in vitro by coculture of both cell types. A major impediment to such studies is the rapid senescence of normal stromal cells. To circumvent this problem, we tested whether human endometrial stromal cells immortalized by expressing a transduced human telomerase reverse transcriptase (TERT) subunit retained the ability to mediate hormonal control of epithelial proliferation in the coculture assay. We found that the telomerized stromal cells were very similar to the parental strain from which they were derived according to criteria of proliferation, karyotype, cellular localization of cytoskeletal markers and nuclear staining, and basal gene expression based on microarray analysis. We also showed that expression of estrogen and progesterone receptors, as assessed by immunodetection, was similar in both telomerized and parental stromal cells. Importantly, the telomerized stromal cells were shown in coculture assay to be as effective as normal stromal cells in regulating the proliferation of endometrial epithelial cells in response to estrogen or progesterone. The availability of these long-lived stromal cells may advance studies addressing the mechanistic, regulatory, and cell structural basis of stromal-epithelial interactions and hormonal responses in normal, preneoplastic, and neoplastic human endometrial tissue.