A comparison of the potential for acute cardiopulmonary adverse effects in dogs during continuous veno-venous hemofiltration with accusol 35 solution with and without induced calcium carbonate particles.

BACKGROUND: Baxter received reports of visible precipitate, identified as calcium carbonate, forming during hemofiltration with Accusol 35 solution. AIM: To evaluate the potential for acute cardiopulmonary adverse effects of Accusol 35 containing exaggerated calcium carbonate particles. METHODS: Anesthetized dogs underwent continuous veno-venous hemofiltration (CVVH) with Accusol 35 containing visible and subvisible particles (≥10 µm) 36 times higher than the maximum concentration specified in the European Pharmacopoeia (P-Accusol), or Accusol 35 conforming to specification (Accusol). Select cardiovascular and blood gas parameters were evaluated during CVVH. Lung tissue samples were collected following CVVH. RESULTS: No differences were observed in cardiovascular and blood gas parameters or lung histology between P-Accusol and Accusol. CONCLUSION: Accusol 35 containing visible and subvisible particles (≥10 µm) 36 times higher than the maximum concentration specified in the European Pharmacopoeia resulted in no acute cardiopulmonary adverse effects compared with Accusol 35 containing no visible particles and subvisible particles within European Pharmacopoeia specification.

Nuclear receptor coactivator-6 attenuates uterine estrogen sensitivity to permit embryo implantation.

Uterine receptivity to embryo implantation is coordinately regulated by 17ß-estradiol (E(2)) and progesterone (P(4)). Although increased E(2) sensitivity causes infertility, the mechanisms underlying the modulation of E(2) sensitivity are unknown. We show that nuclear receptor coactivator-6 (NCOA6), a reported coactivator for estrogen receptor α (ERα), actually attenuates E(2) sensitivity to determine uterine receptivity to embryo implantation under normal physiological conditions. Specifically, conditional knockout of Ncoa6 in uterine epithelial and stromal cells does not decrease, but rather markedly increases E(2) sensitivity, which disrupts embryo implantation and inhibits P(4)-regulated genes and decidual response. NCOA6 enhances ERα ubiquitination and accelerates its degradation, while loss of NCOA6 causes ERα accumulation in stromal cells during the preimplantation period. During the same period, NCOA6 deficiency also caused a failure in downregulation of steroid receptor coactivator-3 (SRC-3), a potent ERα coactivator. Therefore, NCOA6 controls E(2) sensitivity and uterine receptivity by regulating multiple E(2)-signaling components.

Genetic screening reveals an essential role of p27kip1 in restriction of breast cancer progression.

The genetic changes and mechanisms underlying the progression of estrogen-dependent breast cancers to estrogen-independent, antiestrogen-resistant, and metastatic breast cancers are unclear despite being a major problem in endocrine therapy. To identify genes responsible for this progression, we carried out a genetic screening by an enhanced retroviral mutagen (ERM)-mediated random mutagenesis in the estrogen-dependent T47D breast cancer cells. We found that T47D cells contain only one p27kip1 (p27) allele coding for the p27 cyclin-dependent kinase (CDK) inhibitor. An ERM insertion into the p27 locus of T47D cells disrupted the p27 gene and created estrogen-independent and antiestrogen-resistant breast cancer cells that still maintained functional estrogen receptors. Disruption of p27 in T47D cells resulted in several changes, and most of these changes could be rescued by p27 restoration. First, CDK2 activity was increased in the absence of estrogen or in the presence of estrogen antagonists tamoxifen or ICI 182780; second, amplified in breast cancer 1 (AIB1), a cancer overexpressed transcriptional coactivator, was hyperphosphorylated, which made AIB1 a better coactivator for E2F1; and third, growth factor receptor binding protein 2-associated binder 2 (Gab2) and Akt activity were increased following E2F1 overactivation, leading to a significant enhancement of cell migration and invasion. Furthermore, the p27-deficient cells, but not T47D control cells, developed lung metastasis in an ovarian hormone-independent manner when they were i.v. injected into nude mice. In sum, loss of p27 activated AIB1, E2F1, Gab2, and Akt; increased cell migration and invasion; caused antiestrogen insensitivity; and promoted metastasis of breast cancer cells. These findings suggest that p27 plays an essential role in restriction of breast cancer progression.

Haploinsufficiency of the corepressor of estrogen receptor activity (REA) enhances estrogen receptor function in the mammary gland.

Estrogen receptor (ER)-mediated gene expression plays an essential role in mammary gland morphogenesis, function, and carcinogenesis. The repressor of ER activity (REA) is an ER-interactive protein that counterbalances estrogen-induced ER transcriptional activity. Our previous study showed that genetic deletion of both REA alleles resulted in embryonic lethality. This study demonstrates that REA and ERalpha are coexpressed in mammary epithelial cells. REA heterozygous (REA(+/-)) mutant mice exhibit faster mammary ductal elongation in virgin animals, increased lobuloalveolar development during pregnancy, and delayed mammary gland involution after weaning. These morphological phenotypes of REA(+/-) mice are associated with significantly increased cell proliferation and ER transcriptional activities, as indicated by the estrogen response element (ERE)-luciferase reporter in the WT/ERE-Luc and REA(+/-)/ERE-Luc bigenic mice and by the higher expression levels of estrogen-responsive genes such as progesterone receptor and cyclin D1 in the mammary gland. Our analysis also revealed that REA is an important repressor of ER transcriptional activity in the mammary gland under natural, as well as ovariectomized and estrogen-replaced, hormonal conditions. Our results indicate that REA is a physiological modulator of ER function in the mammary gland and that its correct gene dosage is required for maintenance of normal ER activity and normal mammary gland development. Consequently, a reduction or loss of REA function may cause overactivation of ER and increase breast cancer risk in humans.

Stimulation of steroid receptor coactivator-3 (SRC-3) gene overexpression by a positive regulatory loop of E2F1 and SRC-3.

Steroid receptor coactivator 3 (SRC-3, amplified in breast cancer 1, or ACTR) is a transcriptional coactivator for nuclear receptors and certain other transcription factors such as E2F1. SRC-3 is overexpressed in breast cancers, and its overexpression is sufficient to cause mammary carcinomas in vivo. However, the mechanisms controlling endogenous SRC-3 overexpression are unknown. In this study, we identified the first exon and analyzed the 5' regulatory sequence of the SRC-3 gene. We found three evolutionarily conserved regions (ECRs) in the 5' SRC-3 regulatory sequence, and ECR2 makes a major contribution to the SRC-3 promoter activity. The ECR2 region (bp -250/+350) contains several specificity protein 1 (Sp1) binding sites and two E2F1 binding sites. We show that E2F1 can significantly activate the ECR2 promoter activity in a dose-dependent manner. Furthermore, overexpression of E2F1 significantly increases the promoter activity of the endogenous SRC-3 gene and boosts SRC-3 expression in vivo. Conversely, knockdown of E2F1 reduces SRC-3 expression. We demonstrate that the mechanism of E2F1 activity on SRC-3 promoter is independent of the E2F binding sites but relies on the Sp1 element located at bp +150/+160. Sp1, E2F1, and SRC-3 are specifically recruited to this Sp1 site and the interaction between E2F1 and Sp1 is essential to modulate SRC-3 expression. Moreover, SRC-3 coactivates E2F1 activity and thereby additively stimulates a further increase in SRC-3 expression in vivo. These results suggest that in cells with hyperactive E2F1, such as the case encountered in breast cancer cells, there is a positive feedback regulatory loop consisting of E2F1 and SRC-3 to maintain high levels of SRC-3 and E2F1 activity, which may partially interpret the oncogenic role of SRC-3 overexpression.

Estrogen receptor alpha, a molecular switch converting transforming growth factor-alpha-mediated proliferation into differentiation in neuroblastoma cells.

Transforming growth factor-alpha (TGF-alpha) is known to promote both proliferation and differentiation of neural cell progenitors. Using the human neuroblastoma cell line SK-N-BE that is induced to proliferate by TGF-alpha, we demonstrated that the expression of a single transcription factor, the estrogen receptor-alpha (ER alpha), can reroute the TGF-alpha mitogenic signaling toward a path leading to differentiation. With selected mutations in ER alpha and signal transducer and activator of transcription 3 (Stat3), we demonstrated that the blockade of TGF-alpha mitotic potential was not dependent on ER alpha DNA binding activity but required a transcriptionally active Stat3. In neuroblastoma cells, 17 beta-estradiol treatment induced a transient increase in the transcription of estrogen-responsive element-containing promoters including those regulating TGF-alpha and prothymosin alpha synthesis. Based on the data presented, we hypothesized that in the presence of prothymosin alpha, ER alpha activates its direct target genes and increases cell proliferation, whereas in the presence of high levels of TGF-alpha, ER alpha preferentially interacts with Stat3 and causes cell differentiation. Our results reveal a novel form of "end-product" regulation of an intracellular receptor that occurs through recruitment of membrane receptors and their signaling effector system. Cross-coupling between membrane and intracellular receptors has been described by several laboratories. This study proves the relevance of these interactions in cellular responses to growth factors.

In vivo imaging of transcriptionally active estrogen receptors.

Through intracellular receptors, estrogens control growth, differentiation and function of not only reproductive tissues, but also other systems. Estrogen receptors are ligand-dependent transcription factors whose activity is modulated either by estrogens, or by alternative intracellular signaling pathways downstream of growth factors and neurotransmitters. To determine the dynamics of estrogen receptor activity and the dependence of estrogen receptor on 17beta-estradiol in vivo, we generated a transgenic mouse that expresses a luciferase reporter gene under the control of activated estrogen receptors. As expected, luciferase activity, monitored with a cooled charged coupled device camera, paralleled circulating estrogen levels in reproductive tissues and in liver, indicating that the peak transcriptional activity of the estrogen receptor occurred at proestrus. In contrast, in tissues such as bone and brain, the peak activity of estrogen receptors was observed at diestrus. These tissue-specific responses are masked when mice undergo conventional hormone treatment. We also demonstrate that estrogen receptors are active in immature mice before gonadal production of sex hormones as well as in ovariectomized adult mice. These findings emphasize the importance of hormone-independent activation of the estrogen receptor, and have implications for the therapeutic use of estrogens, such as hormone replacement therapy.