Polydimethylsiloxane (PDMS) has become a staple of the microfluidics community by virtue of its simple fabrication process and material attributes, such as gas permeability, optical transparency, and flexibility. As microfluidic systems are put toward biological problems and increasingly utilized as cell culture platforms, the material properties of PDMS must be considered in a biological context. Two properties of PDMS were addressed in this study: the leaching of uncured oligomers from the polymer network into microchannel media, and the absorption of small, hydrophobic molecules (i.e. estrogen) from serum-containing media into the polymer bulk. Uncured PDMS oligomers were detectable via MALDI-MS in microchannel media both before and after Soxhlet extraction of PDMS devices in ethanol. Additionally, PDMS oligomers were identified in the plasma membranes of NMuMG cells cultured in PDMS microchannels for 24 hours. Cells cultured in extracted microchannels also contained a detectable amount of uncured PDMS. It was shown that MCF-7 cells seeded directly on PDMS inserts were responsive to hydrophilic prolactin but not hydrophobic estrogen, reflecting its specificity for absorbing small, hydrophobic molecules; and the presence of PDMS floating in wells significantly reduced cellular response to estrogen in a serum-dependent manner. Quantification of estrogen via ELISA revealed that microchannel estrogen partitioned rapidly into the surrounding PDMS to a ratio of approximately 9:1. Pretreatments such as blocking with serum or pre-absorbing estrogen for 24 hours did not affect estrogen loss from PDMS-based microchannels. These findings highlight the importance of careful consideration of culture system properties when determining an appropriate environment for biological experiments.

The extraction and purification of nucleic acids from complex samples (e.g. blood, biopsied tissue, cultured cells, food) is an essential prerequisite for many applications in biology including genotyping, transcriptional analysis, systems biology, epigenetic analysis, and virus/bacterial detection. In this report, we describe a new process of nucleic acid extraction that utilizes “pinned” aqueous/organic liquid interfaces in microchannels to streamline the extraction mechanism, replacing all washing steps with a single traverse of an immiscible fluid barrier, termed Immiscible Filtration Assisted by Surface Tension (IFAST). Nucleic acids in biological samples are bound to paramagnetic particles and then drawn across the IFAST device (or array of IFAST devices) using a magnet. While the strength of the IFAST barrier is suitable for separation of nucleic acids from lysate in its current embodiment, its permeability can be selectively adapted by adjusting the surface tensions/energies associated with the cell lysate, the immiscible phase, and the device surface, enabling future expansion to other non-nucleic acid applications. Importantly, processing time is reduced from 15–45 minutes to less than 5 minutes while maintaining purity, yield, and scalability equal to or better than prevailing methods. Operation is extremely simple and no additional lab infrastructure is required. The IFAST technology thus significantly enhances researchers’ abilities to isolate and analyze nucleic acids, a process which is critical and ubiquitous in an extensive array of scientific fields.

Regulation of estrogen receptor (ER) concentration is a key component in limiting estrogen responsiveness in target cells. Yet the mechanisms governing ER concentration in the lactotrope cells of the anterior pituitary, a major site of estrogen action, are undetermined. In this study, we used a lactotrope cell line, PR1, to explore regulation of ER protein by estrogen. Estrogen treatment resulted in an approximate 60% decrease in ER steady state protein levels. Suprisingly, the decline in ER protein was apparent within 1 h of estrogen treatment and occurred in the absence of protein synthesis and transcription. Direct regulation of ER protein was further confirmed by pulse chase analysis, which showed that ER protein half-life was shortened from greater than 3 h to 1 h in the presence of estrogen. The estrogen-induced degradation of ER protein could be prevented by pretreatment with peptide aldehyde inhibitors of proteasome protease whereas inhibitors of calpain and lysosomal proteases were ineffective. Inhibition of proteasome activity maintained ER protein at a level equivalent to control cells not stimulated with estrogen but increased estrogen-binding activity by 1.75-fold. Proteolytic regulation of ER by the proteasome is not limited to pituitary lactotrope cells but is also operational in MCF-7 breast cancer cells, suggesting that this may be a common regulatory pathway used by estrogen. These studies describe a nongenomic action of estrogen that involves nuclear ER: rapid proteolysis of ER protein via a proteasome-mediated pathway.

Estrogen receptor alpha (ER␣), a key driver of growth in the majority of breast cancers, contains an unstructured transactivation domain (AF1) in its N terminus that is a convergence point for growth factor and hormonal activation. This domain is controlled by phosphorylation, but how phosphorylation impacts AF1 structure and function is unclear. We found that serine 118 (S118) phosphorylation of the ER␣ AF1 region in response to estrogen (agonist), tamoxifen (antagonist), and growth factors results in recruitment of the peptidyl prolyl cis/trans isomerase Pin1. Phosphorylation of S118 is critical for Pin1 binding, and mutation of S118 to alanine prevents this association. Importantly, Pin1 isomerizes the serine118-proline119 bond from a cis to trans isomer, with a concomitant increase in AF1 transcriptional activity. Pin1 overexpression promotes ligand-independent and tamoxifeninducible activity of ER␣ and growth of tamoxifen-resistant breast cancer cells. Pin1 expression correlates with proliferation in ER␣-positive rat mammary tumors. These results establish phosphorylation-coupled proline isomerization as a mechanism modulating AF1 functional activity and provide insight into the role of a conformational switch in the functional regulation of the intrinsically disordered transactivation domain of ER␣.E strogen receptor alpha (ER␣), a member of the nuclear receptor superfamily of transcription factors, mediates the actions of estrogen in normal physiology and disease (17). ER␣ is expressed in the normal mammary gland and in 70% of human breast cancers and is a key driver of breast cell proliferation (16,26,83). Directed overexpression of ER␣ in the mammary gland is sufficient to induce hyperplasia, and blockade of ER␣ activity by hormonal therapies (aromatase inhibitors, tamoxifen, and fulvestrant) reduces recurrence and improves clinical outcomes of ER␣-positive breast cancer patients (19,22). Two activation functions mediate the transcriptional activity of ER␣, a C-terminal liganddependent AF2 and an N-terminal ligand-independent AF1 (89). Regulation of ER␣ activity via the C-terminal AF2 has been wellcharacterized through biochemical and crystallographic studies and forms the basis for our understanding of hormonal therapy for breast cancer (10,34,84). In the canonical activation pathway, ligand binding initiates C-terminal structural rearrangements that facilitate downstream events, including dimerization, DNA binding, and coregulator interactions, ultimately engaging the basal transcriptional machinery to regulate gene expression. However, ER␣ can also be activated by growth factors and kinases, which phosphorylate the receptor N terminus and other domains to regulate transcription in the absence of direct ligand engagement (for reviews, see references 27, 47, 75, 94, and 95). In contrast to the C-terminal AF2 domain, biochemical and structural mechanisms that control N-terminal AF1 remain poorly understood (48,91).Multiple challenges have hindered molecular dissection of AF1 regulation. First, AF1 resid...

Development of the seminal vesicle (SV) is elicited by androgens and is dependent on epithelialmesenchymal interactions. Androgenic signal trnsmission from the androgen-receptor-positive mesenchyme to the epithellum has been postulated to involve paracrine factors. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is produced by stromal/mesenchl cells and acts s ally on epithal cells. Seminal vesicles (SVs) are male sex-accessory glands derived from the caudal regions of the Wolffian ducts. The development and maintenance of the SV are dependent on epithelial-mesenchymal interactions that are directed by androgens (1-4). SV morphogenesis begins on embryonic day 15 in mice (5) subsequent to the onset of testosterone production by the fetal testes (6). Fetal development of the SV is driven by androgens acting directly on the mesenchyme that induces epithelial development. The mesenchyme must initially mediate the inductive action of androgens because it is the only source of androgen receptor until day 1 postnatally, when epithelial androgen receptors are first detected (7). This conclusion is reinforced by analysis of SV inductions employing normal androgen receptor-positive SV mesenchyme and androgen receptor-negative epithelium (testicular feminization) (8). Dependence on epithelial-mesenchymal interaction continues into adulthood, as demonstrated by homotypic and heterotypic recombination experiments. SV mesenchyme is capable of inducing SV differentiation, morphologically and functionally, from epithelia isolated from any portion of the fetal Wolffian duct (3, 4), adult epididymis (9), and adult ureter and adult ductus deferens (2). Thus, both prenatal and postnatal development depends on instructive signals from the mesenchyme to the epithelium.The identification of paracrine signals that mediate these cell-cell interactions has proven elusive. Recently, a growth factor whose properties are consistent with such an effector has been identified and molecularly cloned. This factor, designated keratinocyte growth factor (KGF), is a heparinbinding growth factor and member of the fibroblast growth factor (FGF) family (FGF-7) (10). KGF is secreted by mesenchymal cells and appears to act solely on epithelial cells (11,12). The present studies were undertaken to investigate the possibility that KOF is an important mediator of the epithelial-mesenchymal interactions required for androgendependent SV development. MATERIALS AND METHODSAnimals and Orgn Culte. SVs were dissected from newborn BALB/c mice obtained from either the Cancer Research Laboratory (University of California, Berkeley) or Simonsen Laboratories (Gilroy, CA). SVs were grown in an organ culture system as described (13). Briefly, the organs were placed on Millipore filters supported on tangula metal grids in 35-mm Petri dishes (Falcon). A basal medium of Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12, 1:1 (vol/vol), supplemented with insulin (10 pg/ml), transferrin (10 ,ug/ml), cholera toxin (10 ng/ml), bov...

The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor ␣ (ER␣) is also subjected to rapid ubiquitinproteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ER␣ proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ER␣ and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ER␣. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ER␣ are mechanistically separable functions of ER␣. We find that proteolysis of ER␣ correlates with the ability of ER␣ mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligandinduced receptor degradation which permits the uncoupling of estrogen-regulated ER␣ proteolysis and transcription.The ubiquitin-proteasome pathway contributes to the control of transcription through the ubiquitination and regulated degradation of multiple components of the transcriptional machinery (10, 34). Among these components is a large list of transactivators whose activity can be related to their proteolytic degradation. For many, particularly those that possess acidic activation domains, such as VP16 and c-myc, sequence elements essential for proteasome-mediated proteolysis reside within transactivation domains (33,43). Several members of the nuclear receptor superfamily are substrates for the ubiquitin-proteasome pathway (11,19,21,24,26,29,37,50,52,55), the first identified being estrogen receptor ␣ (ER␣) (1, 13, 36). ER␣ possesses two transactivation domains, AF-1 and AF-2, which reside in the N terminus and C terminus of the receptor, respectively. These activation domains are bridged by a conserved DNA binding domain and a hinge region responsible for receptor nuclear localization.The transcriptional activity of AF-2 is strictly ligand dependent, but the AF-1 is not; thus, AF-2 received much attention for analysis of the relationship between estrogen-stimulated proteolysis and transcription. AF-2 is highly structured, consisting of 12 ␣-helices that adopt an active conformation upon agonist binding, which exposes a hydrophobic surface where coactivator proteins bind (6). It has been shown that mutations of residues critical for AF-2-mediated transactivation disrupt proteolysis (27, 53). E6-AP, a ubiquitin ligase (35), and the TRIP1/Rpt6/SUG1 (28, 42) subunit of the 19S regulatory cap of the proteasome bind to ER␣ through the coactiva...

Gene expression results from the coordinated actions of transcription factor proteins and coregulators. Estrogen receptor alpha (ER␣) is a ligand-activated transcription factor that can both activate and repress the expression of genes. Activation of transcription by estrogen-bound ER␣ has been studied in detail, as has antagonist-induced repression, such as that which occurs by tamoxifen. How estrogen-bound ER␣ represses gene transcription remains unclear. In this report, we identify a new mechanism of estrogen-induced transcriptional repression by using the ER␣ gene, ESR1. Upon estrogen treatment, ER␣ is recruited to two sites on ESR1, one distal (ENH1) and the other at the proximal (A) promoter. Coactivator proteins, namely, p300 and AIB1, are found at both ER␣-binding sites. However, recruitment of the Sin3A repressor, loss of RNA polymerase II, and changes in histone modifications occur only at the A promoter. Reduction of Sin3A expression by RNA interference specifically inhibits estrogen-induced repression of ESR1. Furthermore, an estrogen-responsive interaction between Sin3A and ER␣ is identified. These data support a model of repression wherein actions of ER␣ and Sin3A at the proximal promoter can overcome activating signals at distal or proximal sites and ultimately decrease gene expression.Downregulation of receptors by their ligands is a fundamental process by which cells control sensitivity to stimuli. For steroid hormones, this involves lipophilic ligands binding to intracellular receptors to induce a decline in receptor number. Regulation of estrogen (E2) receptor alpha (ER␣) by E2 is one example. The E2-induced decline in ER␣ is, in part, mediated through direct regulation of the protein. It is well documented that decreases in ER␣ protein levels in response to E2 occur via the ubiquitin-proteasome pathway (1, 42). The mRNA levels of ER␣ also decrease, but the mechanism responsible for E2-induced repression of the ER␣ gene, ESR1, is not established (5, 49, 52).ER␣ is a ligand-activated transcription factor that mediates the effects of E2 by regulating gene expression. Activation by ER␣ has been studied in detail, but little is understood about how E2-bound ER␣ represses transcription. E2-induced repression is, however, of significant biological importance. Microarray analyses of E2-treated breast cancer cell lines show that the number of repressed genes is greater than or near the number of activated genes (10,19,29,32). Yet, there are limited reports investigating E2-induced repression, and no generalized mechanism has emerged (6,13,22,25,43,47,59,60,71,74). Antagonistinduced repression by selective ER modulators involves conformational changes that prevent coactivator binding to ER␣ (55). Such a conformational blockade does not occur with agonist binding and thus cannot account for E2-induced gene repression.Many repressive complexes exist to restrict gene expression in response to cellular signals. One example is the Sin3 complex, which was identified in yeast but is conserved in mammals (41, ...

Estrogens promote cell proliferation and metastases in several human cancers. Here, we describe a different action of estrogens likely to contribute to tumor development-blocking immunosurveillance. In breast cancer cells, increasing concentrations of estrogen induce increasing levels of the granzyme B inhibitor, SerpinB9/proteinase inhibitor 9 (PI-9) and progressively block cell death induced by NK92 natural killer (NK) cells, but do not block killing by a second NK cell line, NKL cells. RNA interference knockdown of PI-9 abolishes estrogen's ability to block NK92 cell-induced cytotoxicity. Expressing elevated levels of estrogen receptor a (ERa) increases the induced level of PI-9, and makes tamoxifen (TAM), but not raloxifene or ICI 182,780, a potent inducer of PI-9. At elevated levels of ERa, induction of PI-9 by estradiol or TAM blocks killing by both NK92 and NKL cells. When the Erk pathway is activated with epidermal growth factor, the concentration of estrogen required to induce a protective level of PI-9 is reduced to 10 pM. Elevated concentrations of estrogen and ER may provide a dual selective advantage to breast cancer cells by controlling PI-9 levels and thereby blocking immunosurveillance. Expressing elevated levels of ERa reveals a potentially important difference in the effects of TAM, raloxifene and ICI 182,780 on immunosurveillance in breast cancer.