TFAP2C governs the luminal epithelial phenotype in mammary development and carcinogenesis.

Molecular subtypes of breast cancer are characterized by distinct patterns of gene expression that are predictive of outcome and response to therapy. The luminal breast cancer subtypes are defined by the expression of estrogen receptor-alpha (ERα)-associated genes, many of which are directly responsive to the transcription factor activator protein 2C (TFAP2C). TFAP2C participates in a gene regulatory network controlling cell growth and differentiation during ectodermal development and regulating ESR1/ERα and other luminal cell-associated genes in breast cancer. TFAP2C has been established as a prognostic factor in human breast cancer, however, its role in the establishment and maintenance of the luminal cell phenotype during carcinogenesis and mammary gland development have remained elusive. Herein, we demonstrate a critical role for TFAP2C in maintaining the luminal phenotype in human breast cancer and in influencing the luminal cell phenotype during normal mammary development. Knockdown of TFAP2C in luminal breast carcinoma cells induced epithelial-mesenchymal transition with morphological and phenotypic changes characterized by a loss of luminal-associated gene expression and a concomitant gain of basal-associated gene expression. Conditional knockout of the mouse homolog of TFAP2C, Tcfap2c, in mouse mammary epithelium driven by MMTV-Cre promoted aberrant growth of the mammary tree leading to a reduction in the CD24(hi)/CD49f(mid) luminal cell population and concomitant gain of the CD24(mid)/CD49f(hi) basal cell population at maturity. Our results establish TFAP2C as a key transcriptional regulator for maintaining the luminal phenotype in human breast carcinoma. Furthermore, Tcfap2c influences development of the luminal cell type during mammary development. The data suggest that TFAP2C has an important role in regulated luminal-specific genes and may be a viable therapeutic target in breast cancer.

Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123.

To detect intracellular oxidant formation during reoxygenation of anoxic endothelium, the oxidant-sensing fluorescent probes, 2',7'-dichlorodihydrofluorescein diacetate, dihydrorhodamine 123, or 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate were added to human umbilical vein endothelial cells during reoxygenation. None of these fluorescent probes were able to differentiate the controls from the reoxygenated cells in the confocal microscope. However, dihydrofluorescein diacetate demonstrated fluorescence of linear structures, consistent with mitochondria, in reoxygenated endothelium. This work tests the hypothesis that dihydrofluorescein diacetate is a better fluorescent probe for detecting intracellular oxidants because it is more reactive toward specific oxidizing species. To investigate this, dihydrofluorescein diacetate was exposed to various oxidizing species (hydrogen peroxide, superoxide [KO2], peroxynitrite, nitric oxide, horseradish peroxidase, ferric iron, xanthine oxidase, cytochrome c, and lipoxygenase) and compared with the three other popular probes. Though oxidized dihydrofluorescein has higher molar fluorescence, comparison of the reactions of dihydrofluorescein with these other three probes in a cell-free system indicates that dihydrofluorescein is sometimes less fluorescent than the other probes. In addition, we find that the reactivity of all of the probes is very complex. Based on the results reported here, it is no longer appropriate to think of these probes as detecting a specific oxidizing species in cells, such as H2O2, but rather as detectors of a broad range of oxidizing reactions that may be increased during intracellular oxidant stress. Cell-loading studies indicate that dihydrofluorescein achieves higher intracellular concentrations than the second brightest intracellular probe, 2',7'-dichlorodihydrofluorescein. This fact and its higher molar fluorescence may account for the superior brightness of dihydrofluorescein diacetate. Dihydrofluorescein diacetate may be a superior fluorescent probe for many cell-based studies.

Extracellular iron (II) can protect cells from hydrogen peroxide.

We hypothesized that exposure of cells to H2O2 plus Fe2+ would increase formation of cell-derived lipid peroxides that would inactivate prostaglandin H synthase, resulting in decreased prostaglandin synthesis. Therefore, we treated human endothelial cells with 0-100 microM H2O2 followed immediately by addition of 0-200 microM Fe2+. After oxidant exposure, cells were stimulated with 20 microM arachidonic acid to induce prostaglandin I2 (PGI2) synthesis. Adding 100 microM H2O2 prior to arachidonic acid decreased PGI2 synthesis more than 80%. However, to our surprise, the addition of Fe2+, in increasing amounts, progressively protected PGI2 synthesis against the harmful effects of H2O2. A ratio of one part H2O2 to two parts Fe2+ offered almost complete protection, whereas Fe3+ did not protect PGI2 synthesis from H2O2. We found that 100 microM H2O2 was not cytolytic; however, 250 microM H2O2 was cytolytic; Fe2+ protected against this cytotoxicity. In addition, extracellular Fe2+ prevented the rise in intracellular calcium caused by H2O2 and extracellular Fe2+ preserved intracellular glutathione in H2O2-exposed cells. Electron paramagnetic resonance spin trapping demonstrated that extracellular Fe2+ generated the hydroxyl free radical, HO. outside the cell. We speculate that extracellular Fe2+ protects the intracellular space from H2O2 by initiating the Fenton reaction outside the cell. This reductive cleavage of H2O2 generates HO. in the extracellular space, where much of the HO. will react with noncellular components, thereby protecting the cell interior.