Physical and functional interactions between Cas and c-Src induce tamoxifen resistance of breast cancer cells through pathways involving epidermal growth factor receptor and signal transducer and activator of transcription 5b.

High expression of the adaptor molecule Cas has been linked to resistance to the antiestrogen tamoxifen, both in tissue culture and in human tumors. The aim of this study was to elucidate the mechanism(s) by which overexpression of Cas confers resistance to tamoxifen. Cas overexpression in MCF-7 breast cancer cells was shown to alleviate both tamoxifen-mediated growth inhibition and induction of apoptosis. This enhancement of cell proliferation/survival occurred in the absence of detectable effects on estrogen receptor (ER) transcriptional activity under conditions where tamoxifen was present, indicating that Cas-dependent tamoxifen resistance is not the result of a switch to an ER-negative phenotype or enhanced responses to the partial agonist activity of tamoxifen. Instead, we present evidence, suggesting that Cas promotes tamoxifen resistance by deregulation of alternative cell proliferation pathways, particularly those mediated through enhanced c-Src protein tyrosine kinase activity arising from Cas/c-Src interactions. Overexpression of Cas was found to drive endogenous c-Src into complex with Cas, a process that has been shown previously to cause up-regulation of c-Src tyrosine kinase activity. MCF-7 cells overexpressing Cas exhibited increased phosphorylation of two c-Src substrates, Tyr845 in the kinase domain of the epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription (STAT) 5b. Importantly, Cas-dependent protection from the antiproliferative effects of tamoxifen was reversed by the expression of dominant inhibitory variants of these substrates (Y845F EGFR and COOH-terminally truncated STAT5b). Based on these findings, we suggest that the Cas/c-Src/EGFR/STAT5 signaling axis is a major regulator of tamoxifen-resistant breast cancer cell growth and survival.

Remarkable differences in the biochemical fate of Cd2+, Hg2+, CH3Hg+ and thimerosal in red blood cell lysate.

Humans are environmentally exposed to potentially toxic Cd and Hg species and to the Hg compound thimerosal (THI), an antibactericidal vaccine additive. Previous studies have revealed that Cd2+, Hg2+ and CH3Hg+ are taken up by red blood cells (RBCs) and bind to cytosolic glutathione (GSH) and/or hemoglobin (Hb). Since interactions in the cytosol of RBCs may be linked to their hemolysis, a more comprehensive characterization of these interactions was sought. After the addition of each Cd and Hg species to RBC lysate, the mixtures were analyzed after 5 min, 2 h and 6 h by size-exclusion chromatography (SEC) coupled on-line to an inductively coupled plasma atomic emission spectrometer (ICP-AES). In contrast to previous studies, however, reducing conditions were maintained by employing a 100 mM Tris buffer mobile phase (pH 7.4), which contained ∼2.5 mM of glutathione (GSH). At ≥2 h, ∼85% of Cd2+ weakly interacted with hemoglobin (Hb), while ∼13% eluted as (GS)xCd and ∼2% bound to a ≥70 kDa Cd-binding protein. In contrast, ∼6% of Hg2+ co-eluted with Hb at all time points, while ∼94% eluted as (GS)xHg. The results for CH3Hg+ showed that ∼5% of Hg co-eluted with Hb, while for THI this percentage gradually increased to 12% (6 h). The remaining Hg eluted as GS-HgCH3 and GS-HgCH2CH3. Our results revealed remarkable differences in the interaction of the investigated Cd and Hg species with cytosolic RBC constituents. The formation of (Hb)xHg species, regardless of which Hg compound was added, suggests their mammalian toxicology to be intertwined with the metabolism of Fe.

Stimulation of the novel estrogen receptor-alpha intronic TERP-1 promoter by estrogens, androgen, pituitary adenylate cyclase-activating peptide, and forskolin, and autoregulation by TERP-1 protein.

The estrogen receptor-alpha (ERalpha) pituitary-specific variant, TERP-1, is regulated dramatically by physiological status. We examined hormonal regulation of the TERP-1 promoter in transient transfection assays in GH3 somatolactotrope cells. We found that 17beta-estradiol (E2), genistein, androgen, pituitary adenylate cyclase-activating peptide, and forskolin (FSK) all stimulated TERP-1 promoter activity, whereas progesterone had no effect. ERalpha bound to a palindromic estrogen response element (ERE) and two half-site EREs; mutation of any of these sites decreased basal expression and completely obliterated E2 stimulation. In contrast, mutation of an activator protein-1 site decreased basal and FSK-stimulated promoter activity, but not E2 or androgen stimulation. The pure antiestrogen ICI 182,780 suppressed E2 and genistein, but not FSK or androgen, stimulation. Similarly, mutation of the ERE palindrome or half-site EREs suppressed promoter stimulation by E2 and genistein, but not by androgen or FSK. Because TERP-1 levels regulate ERalpha function on model promoters, we tested TERP-1 modulation of its own and other physiological promoters. TERP-1 suppressed basal and E2-stimulated expression of its own promoter. TERP-1 suppression required the ERE regions of the promoter, and the dimerization domain of TERP-1. TERP-1 overexpression also suppressed E2 stimulation of the progesterone receptor and prolactin promoters. Thus, estrogens, androgen, and FSK can stimulate TERP-1 promoter activity, and increased TERP-1 expression modulates E2 stimulation of physiological promoters. These data suggest that TERP-1 regulation may play a significant role in modifying pituitary ERalpha responses.

Estrogen negatively regulates epidermal growth factor (EGF)-mediated signal transducer and activator of transcription 5 signaling in human EGF family receptor-overexpressing breast cancer cells.

Breast cancer cell growth may be stimulated by 17beta-estradiol (E2) or growth factors like epidermal growth factor (EGF). However, tumors typically depend on only one of these pathways and may overexpress either estrogen receptor (ER) or EGF receptor (EGFR) and related family members. Tumors overexpressing EGFR are more aggressive than those expressing ER. Intracellular mediators of these growth-stimulatory pathways are not completely defined, but one potential common mediator of EGF and E2 signaling is the transcription factor signal transducer and activator of transcription 5 (STAT5). To investigate the role of STAT5 in potential crosstalk between E2 and EGF, MDA-MB231 and SKBr3 breast cancer cells, which are ER-negative and overexpress human EGF family receptors, were used. Introduction of ERalpha and treatment with E2 decreased EGF-induced tyrosine phosphorylation of STAT5b, basal and EGF-induced STAT5-mediated transcription, and EGF-stimulated DNA synthesis in these cells. Suppressive effects of E2-EpsilonRalpha were specific for STAT5, as EGF stimulation of MAPK was unaffected. Deletion/mutation analysis of ERalpha demonstrated that the DNA-binding domain was insufficient, and that the ligand-binding domain was required for these responses. ERalpha transcriptional activity was not necessary for suppression of STAT5 activity. Overexpression of c-Src did not prevent suppression of STAT5 activity by E2 and ERalpha. However, ERalpha did prevent basal increases in STAT5 activity with overexpressed c-Src. In the context of human EGF receptor family overexpression, E2-ER opposes EGF signaling by regulating STAT5 activity. STAT5 may be a crucial point of signaling for both E2 and growth factors in breast cancer cells, allowing targeted therapy for many types of breast tumors.

Physiologically relevant plasma d,l-homocysteine concentrations mobilize Cd from human serum albumin.

Although low-level chronic exposure of humans to cadmium (Cd(2+)) can result in a variety of adverse health effects, little is known about the role that its interactions with plasma proteins and small molecular weight (SMW) ligands in the bloodstream may play in delivering this metal to its target organs. To gain insight, a Cd-human serum albumin (HSA) 1:1 (molar ratio) complex was analyzed by size exclusion chromatography (SEC) coupled on-line to a flame atomic absorption spectrometer (FAAS). Using a phosphate buffered saline (PBS)-buffer mobile phase, the stability of the Cd-HSA complex was investigated in the presence of 2.0mM of SMW ligands, including taurine, acetaminophen, l-methionine, l-cysteine (Cys), d,l-homocysteine (hCys) or l-cysteine methyl-ester (Cys-Me). While taurine, acetaminophen and l-methionine did not affect its integrity, Cys, hCys and Cys-Me completely abstracted Cd from HSA. Subsequent investigations into the effect of 1.5, 1.0 and 0.5mM Cys and hCys on the integrity of the Cd-HSA complex revealed clear differences with regard to the nature of the eluting SMW-Cd species between these structurally related endogenous thiols. Interestingly, the Cd-specific chromatograms that were obtained for 0.5mM hCys revealed the elution of an apparent mixture of the parent Cd-HSA complex with a significant contribution of a structurally uncharacterized CdxhCysy species. Since this hCys concentration is encountered in blood plasma of hyperhomocysteinemia patients and since previous studies by others have revealed that a SH-containing carrier mediates the uptake of Cd into hepatocytes, our results suggest that plasma hCys may play a role in the toxicologically relevant translocation of Cd from the bloodstream to mammalian target organs.

Evolution of autologous chondrocyte repair and comparison to other cartilage repair techniques.

UNLABELLED: Articular cartilage defects have been addressed using microfracture, abrasion chondroplasty, or osteochondral grafting, but these strategies do not generate tissue that adequately recapitulates native cartilage. During the past 25 years, promising new strategies using assorted scaffolds and cell sources to induce chondrocyte expansion have emerged. We reviewed the evolution of autologous chondrocyte implantation and compared it to other cartilage repair techniques. METHODS: We searched PubMed from 1949 to 2014 for the keywords "autologous chondrocyte implantation" (ACI) and "cartilage repair" in clinical trials, meta-analyses, and review articles. We analyzed these articles, their bibliographies, our experience, and cartilage regeneration textbooks. RESULTS: Microfracture, abrasion chondroplasty, osteochondral grafting, ACI, and autologous matrix-induced chondrogenesis are distinguishable by cell source (including chondrocytes and stem cells) and associated scaffolds (natural or synthetic, hydrogels or membranes). ACI seems to be as good as, if not better than, microfracture for repairing large chondral defects in a young patient's knee as evaluated by multiple clinical indices and the quality of regenerated tissue. CONCLUSION: Although there is not enough evidence to determine the best repair technique, ACI is the most established cell-based treatment for full-thickness chondral defects in young patients.

Inhibition of the splicing of glucose-6-phosphate dehydrogenase precursor mRNA by polyunsaturated fatty acids.

Polyunsaturated fatty acids inhibit the expression of hepatic glucose-6-phosphate dehydrogenase (G6PD) by changes in the amount of G6PD pre-mRNA in the nucleus in the absence of changes in the transcription rate of the gene. We have compared the nuclear accumulation of partially and fully spliced mRNA for G6PD in the livers of mice fed diets high versus low in polyunsaturated fat. Consumption of a diet high in polyunsaturated fat decreased the accumulation of partially spliced forms of the G6PD pre-mRNA. Examining the fate of multiple introns within the G6PD primary transcript indicated that in mice fed a high fat diet, G6PD pre-mRNA containing intron 11 accumulated within the nucleus, whereas G6PD mature mRNA abundance was inhibited 50% or more within the same livers. Transient transfection of RNA reporters into primary hepatocyte cultures was used to localize the cis-acting RNA element involved in this regulated splicing. Reporter RNA produced from constructs containing exon 12 were decreased in amount by arachidonic acid. The extent of this decrease paralleled that seen in the expression of the endogenous G6PD mRNA. The presence of both exon 12 and a neighboring intron within the G6PD reporter RNA was essential for regulation by polyunsaturated fatty acid. Inhibition was not dependent on the presence of the G6PD polyadenylation signal and the 3'-untranslated region, but substitution with the SV40 poly(A) signal attenuated the inhibition by arachidonic acid. Thus, exon 12 contains a putative splicing regulatory element involved in the inhibition of G6PD expression by polyunsaturated fat.

Nutritional regulation of mRNA processing.

Understanding how a cell adapts to dietary energy in the form of carbohydrate versus energy in the form of triacylglycerol requires knowledge of how the activity of the enzymes involved in lipogenesis is regulated. Changes in the activity of these enzymes are largely caused by changes in the rate at which their proteins are synthesized. Nutrients within the diet can signal these changes either via altering hormone concentrations or via their own unique signal transduction pathways. Most of the lipogenic genes are regulated by changes in the rate of their transcription. Glucose-6-phosphate dehydrogenase (G6PD) is unique in this group of enzymes in that nutritional regulation of its synthesis involves steps exclusively at a posttranscriptional level. G6PD activity is enhanced by the consumption of diets high in carbohydrate and is inhibited by the consumption of polyunsaturated fat. In this review, evidence is presented that changes in the rate of synthesis of the mature G6PD mRNA involves regulation of the efficiency of splicing of the nascent G6PD transcript. Furthermore, this regulation involves the activity of a cis-acting sequence in the G6PD primary transcript. This sequence in exon 12 is essential for the inhibition of G6PD mRNA splicing by PUFA. Understanding the mechanisms by which nutrients alter nuclear posttranscriptional events will provide new information on the breadth of mechanisms involved in gene regulation.