Differential up-regulation of gap junction connexin 26 gene in mammary and uterine tissues: the role of Sp transcription factors.

The mRNA and protein expressions of connexin 26 (Cx26) in rat mammary gland and uterus can be up-regulated during pregnancy as well as by the administration of human CG (hCG). In the present study, we found that the time course and magnitude of Cx26 induction by hCG was different in these two tissues. The molecular mechanism underscoring this difference was therefore investigated. We had previously demonstrated that both Sp1 and Sp3 transcription factors play a functional role in Cx26 expression. By the electrophoretic mobility shift assay, nuclear extracts from both virgin mammary gland and uterus were capable of binding to a labeled oligonucleotide probe that contained the proximal GC box and formed three protein-DNA complexes (C1, C2, and C3). In the mammary gland, pregnancy enhanced the intensity of all three complexes, whereas in the uterine tissue there was a decrease in the C2 and C3 complexes and an emergence of a new major component, C4 complex. In the supershift study, the C1 complex could be supershifted only by an antibody against Sp1, whereas C2, C3, and C4 could all be supershifted by an antibody against Sp3, suggesting a potential presence of Sp3 isoforms of various sizes. We therefore conclude that the basal Sp profiles in virgin mammary gland and uterine tissue are similar. However, in response to pregnancy, the changes in Sp profile are tissue specific and may account for the temporal and quantitative differences between these two tissues in Cx26 induction.

Cyclic AMP modifies the cellular distribution of connexin43 and induces a persistent increase in the junctional permeability of mouse mammary tumor cells.

Direct communication between cells via gap junctions is thought to be an important component of homeostasis and coordinated cellular responses to external signals. We investigated how the second messenger cAMP exerts its effects on junctional communication in a mouse mammary tumor cell line, MMT22. Junctional permeance was quantitatively assessed using dye microinjection and video microscopy. An increase of permeance was found after exposure to 8-bromo-cAMP, being detectable after 30 minutes of treatment and attaining a fourfold higher level of permeance by 24 hours. This elevated level was maintained with continuous exposure to 8-bromo-cAMP for seven days. The permeability change was accompanied by an increase in gap junctions as shown by freeze-fracture electron microscopy and by confocal microscopy using antibodies directed against the gap junction protein, connexin43. The amount of detergent-insoluble connexin43 also increased with 8-bromo-cAMP treatment, and most of the increase could be attributed to an increase of slower migrating (i.e. phosphorylated) species of connexin43. However, connexin43 mRNA and the total cellular content of connexin43 did not change over this period of exposure to 8-bromo-cAMP, as shown by densitometric analyses of northern and western blots. We conclude that 8-bromo-cAMP affects the distribution of connexin43 such that a greater proportion of the protein is utilized for channel formation. Since these changes were relatively slow to develop and persisted with prolonged exposure to 8-bromo-cAMP, it is possible that the junctional permeability of these mammary tumor cells is linked to the 'basal' level of cAMP, i.e. levels maintained by the cells in accordance with a particular cell state.

Measurement of gap junctional communication by fluorescence activated cell sorting.

Cell-to-cell communication via gap junctions has played a fundamental role in the orderly development of multicellular organisms. Current methods for measuring this function apply mostly to homotypic cell populations. The newly introduced Fluorescence Activated Cell Sorting (FACS) method, albeit with some limitations, is simple, reliable, and quantitative in measuring the dye transfer via gap junctions in both homotypic and heterotypic cell populations. In the homotypic setting, the result in dye transfer from the FACS method is comparable to the scrape-loading and microinjection methods. Using this FACS method, we observed a decline of cell-to-cell communication in transformed and cancer cells. We also observed a differential degree of communication between two heterotypic cell populations depending on the direction of dye transfer.

Up-regulation of estrogen receptors by nonsteroidal antiestrogens in human breast cancer.

Development of resistance to hormonal therapy in breast cancer is frequently associated with a decline or loss of cellular estrogen receptors. Agents which up-regulate the receptor may reduce the incidence of hormonal resistance. Antiestrogens at concentrations ranging from 0.1 to 1 microM produced a 2- to 4-fold increase of estrogen receptors in MCF-7 and T-47D breast cancer cells. This increase, which occurred as early as 3 h and was sustained throughout the 4 days of continuous exposure to tamoxifen, was primarily due to an enhancement in receptor synthesis.

Breast cancers negative for estrogen receptor but positive for progesterone receptor, a true entity?

By the conventional steroid-binding assay method for receptor, 3% of 1,095 primary breast cancers (or 10.6% of 263 premenopausal tumors) were classified as negative for estrogen receptor (ER), but positive for progesterone receptor (PR). The true ER status in this rare group of tumors was further investigated by the enzyme-immunoassay (EIA) or immunocytochemical (ICA) staining method using monoclonal antibodies H222 and D547. Immunoreactive ER was present in nine ER-/PR+ tumors studied, whereas it was not detectable in nine age-matched ER-/PR- tumors. Immunoreactive ER was also present in 24 ER+ breast cancers studied, and was particularly higher in tumors that were PR+. Measurement of immunoreactive ER by monoclonal antibody method provides certain advantages over the conventional dextran-coated charcoal (DCC) method, especially in ER-/PR+ tumors.