DNA ploidy of human breast cancer

Ploidy was determined on 663 resectable primary tumors from untreated patients. Nuclei obtained by mechanical disaggregation of frozen tissue were stained with propidium iodide and analysed in a FACS IV. Aneuploidy was detected in 73% of cases. It was not significantly related to nodal involvement or tumor size, although the highest frequencies were observed in large tumors (88%) or with more than 10 positive nodes (77%). Aneuploidy was more frequently observed in ductal infiltrating (81%) than in lobular histology and in tumors lacking both progesterone and estrogen receptors (85%). Analysis of ploidy in primary and synchronous lymph node metastases from the same patient showed a high agreement rate (90%) of DNA patterns simply defined as diploid or aneuploid. However, differences in DNA stemlines and DNA indices between the two synchronous lesions from the same patient were a rather frequent event.     

Effect of sodium butyrate on human breast cancer cell lines

We have investigated the effects exerted by sodium butyrate (NaBu) on the growth and cell cycle perturbations of four human breast cancer cell lines (MCF7, T47D, MDA-MB231 and BT20) with different steroid receptor profiles. Moreover, since one of the supposed mechanisms of action for NaBu activity involves the induction of apoptosis, we have studied the effects of NaBu on DNA fragmentation by agarose gel electrophoresis and flow cytometry. In all investigated cell lines, NaBu exerted a time- and dose-dependent inhibition of growth and caused a maximum inhibitory effect (85% to 90%) at the concentration of 2.5 m m . The inhibition was already evident after 3 days of treatment. The antiproliferative effect of NaBu was associated with a persistent block of cells in the G₂M phase. The block was associated with apoptosis only in oestrogen-receptor positive cell lines. The inhibiting effect of NaBu in hormone-dependent and independent cell lines and its ability to induce apoptosis through a cell cycle perturbation in hormone-dependent cell lines may have important implications in the treatment of human tumours including breast cancer.     

Influence of culture conditions on the estrogenic cell growth stimulation of human breast cancer cells

17 beta-Estradiol is a potent mitogen for hormone-dependent cell lines (MCF-7, T47D and ZR 75.1). However, the degree of hormone sensitivity is very much influenced by culture conditions. In order to understand which factors modulate estrogenic effects on cell growth, four different culture conditions were used: (a) medium with dextran-coated charcoal-treated fetal calf serum (DCC-FCS); (b) medium with dextran-coated charcoal-treated growth factor-inactivated serum (DCC-FCSd); (c) serum-free medium, after a 24-h incubation with serum to allow cell attachment; and (d) serum-free medium on collagen IV-treated plates. In all cell lines the highest cell growth stimulation was achieved when estradiol was added in the presence of 5% DCC-FCS, whereas reducing or removing serum from the culture medium resulted in a decrease in cell proliferation stimulation. We postulate that serum contains some still unknown components able to modulate the degree of estrogenic action in endocrine-dependent breast cancer cell lines.     

Relationship between steroid receptors (as continuous variables) and response to adjuvant treatments in postmenopausal women with node-positive breast cancer.

In current clinical practice for breast cancer patients, estrogen (ER) and progesterone receptor (PgR) concentrations, quantified by the dextran-coated charcoal assay, are categorized by an arbitrary cutoff into a negative or positive status. However, although the results obtained with this approach are easy to interpret, such a representation could oversimplify the relationship between ER and PgR content and patient outcome and imply an assumption of monotonicity, which is generally expected but rarely proven. We evaluated the relationship between ER and PgR content (considered on a continuous scale) and clinical outcome, using a flexible statistical model, in a group of postmenopausal patients with N-positive operable tumors who were submitted to surgery and different adjuvant treatments (tamoxifen or CMF). Univariate analysis indicated that in the tamoxifen-treated group, ER level, number of metastatic nodes (pN) and age, but not PgR, were significant indicators of clinical outcome (p = 0.032, p = 0.021 and p = 0.029, respectively). Multivariate analysis indicated that in this group of patients there was no interaction between variables, and in the final model for disease-free survival (DFS) only ER and pN were retained with an overall predictive ability of the regression model of 0.723, as evaluated by Harrell's c. However, pN markedly contributed to the predictive ability of the model with respect to ER, since a marked decrease in Harrell's c statistic (c = 0.582) was observed when pN was removed from the model. In the CMF-treated group, only pN affected clinical outcome. When the estimated DFS curves obtained from the final Cox regression models were plotted according to four values of ER (in the tamoxifen-treated group) or three values of pN (in the CMF-treated group) we observed that in the tamoxifen-treated group patients with an ER concentration equal to 0 fmol/mg cytosol protein had the worst prognosis, whereas a marked improvement of the expected DFS was observed for patients with a low but detectable ER level (generally classified as ER-negative because falling below the conventional cutoff value of 10 fmol/mg cytosol protein). Our results seem to suggest that the use of steroid receptor concentrations on a continuous scale, instead of dichotomous "status", is to be preferred in the choice of adequate therapeutic strategies.     

Genistein blocks breast cancer cells in the G(2)M phase of the cell cycle

Genistein, a natural isoflavone phytoestrogen present in soybeans, caused a dose-dependent growth inhibition of the two hormone-sensitive cell lines T47D and ZR75.1 and of the two hormone-independent cell lines MDAMB-231 and BT20. Flow cytometric analysis of cells treated for 4 days with 15 and 30 microM genistein showed a dose-dependent accumulation in the G(2)M phase of the cell cycle. At the highest tested concentration, there was a sevenfold increase in the percentage of cells in G(2)M (63%) with respect to the control (9%) in the case of T47D cells and a 2.4-fold increase in the case of BT20. An intermediate fourfold accumulation was observed in the case of MDAMB-231 and ZR75.1. The G(2)M arrest was coupled with a parallel depletion of the G(0)/G(1) phase. To understand the mechanism of action underlying the block in G(2)M induced by genistein, we investigated the expression and the activity of cyclins and of cyclin-dependent kinases specifically involved in the G(2)-->M transition. As expected, p34(cdc-2) expression, monitored by Western blotting, was unaffected by genistein treatment in all cell lines. With exception of the T47D cell line, we revealed an increase in the tyrosine phosphorylated form of p34, suggesting an inactivation of the p34(cdc-2) catalytic activity consequent to treatment of cells with genistein. In fact, immunoprecipitates from genistein-treated MDAMB-231 and BT20 cells displayed a fourfold decrease in kinase activity evaluated using the histone H1 as substrate. Conversely, no variation in kinase activity was observed between treated and untreated ZR75.1 cells despite the increase in p34 phosphorylation. In cells treated with 30 microM genistein, cyclin B(1) (p62) increased 2.8-,8-and 103-fold, respectively, in BT20, MDAMB-231, and ZR75.1 cells, suggesting an accumulation of the p62, which is instead rapidly degraded in cycling cells. No effects were observed on cyclin expression in T47D cells. We therefore conclude that genistein causes a G(2)M arrest in breast cancer cell lines, but that such growth arrest is not necessarily coupled with deregulation of the p34(cdc-2)/cyclin B(1) complex only in all of the studied cell lines.