Reporter gene assays (RGAs) are commonly used to measure biological pathway modulation by small molecules. Understanding how such compounds interact with the reporter enzyme is critical to accurately interpret RGA results. To improve our understanding of reporter enzymes and to develop optimal RGA systems, we investigated eight reporter enzymes differing in brightness, emission spectrum, stability, and substrate requirements. These included common reporter enzymes such as firefly luciferase (Photinus pyralis), Renilla reniformis luciferase, and β-lactamase, as well as mutated forms of R. reniformis luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and a proprietary luciferase termed "NanoLuc" derived from the luminescent sea shrimp Oplophorus gracilirostris. To determine hit rates and structure-activity relationships, we screened a collection of 42,460 PubChem compounds at 10 μM using purified enzyme preparations. We then compared hit rates and chemotypes of actives for each enzyme. The hit rates ranged from <0.1% for β-lactamase to as high as 10% for mutated forms of Renilla luciferase. Related luciferases such as Renilla luciferase mutants showed high degrees of inhibitor overlap (40-70%), while unrelated luciferases such as firefly luciferases, Gaussia luciferase, and NanoLuc showed <10% overlap. Examination of representative inhibitors in cell-based assays revealed that inhibitor-based enzyme stabilization can lead to increases in bioluminescent signal for firefly luciferase, Renilla luciferase, and NanoLuc, with shorter half-life reporters showing increased activation responses. From this study we suggest strategies to improve the construction and interpretation of assays employing these reporter enzymes.

Breast cancer development is a complex pathobiological process involving sequential genetic alterations in normal epithelial cells that results in uncontrolled growth in a permissive microenvironment. Accordingly, physiologically relevant models of human breast cancer that recapitulate these events are needed to study cancer biology and evaluate therapeutic agents. Here, we report the generation and utilization of the human breast cancer in mouse (HIM) model, which is composed of genetically engineered primary human breast epithelial organoids and activated human breast stromal cells. By using this approach, we have defined key genetic events required to drive the development of human preneoplastic lesions as well as invasive adenocarcinomas that are histologically similar to those in patients. Tumor development in the HIM model proceeds through defined histological stages of hyperplasia, DCIS to invasive carcinoma. Moreover, HIM tumors display characteristic responses to targeted therapies, such as HER2 inhibitors, further validating the utility of these models in preclinical compound testing. The HIM model is an experimentally tractable human in vivo system that holds great potential for advancing our basic understanding of cancer biology and for the discovery and testing of targeted therapies.cancer model ͉ human in mouse ͉ tissue reconstitution

Previous studies have shown that human fetal adrenal gland from 17-to 20-week-old fetuses expressed pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, which were localized on chromaffin cells. Pituitary adenylate cyclase-activating polypeptide is a 38-residue ␣-amidated neuropeptide (PACAP-38) 1 originally isolated from the ovine hypothalamus for its ability to stimulate cAMP formation in rat anterior pituitary cells. Processing of PACAP-38 can generate a 27-amino acid amidated peptide (PACAP-27) that exhibits 68% sequence identity with vasoactive intestinal polypeptide (VIP), thus identifying PACAP as a member of the VIP/secretin/glucagon superfamily of regulatory peptides (1, 2).The effects of PACAP are mediated through interaction with two types of high affinity receptors: type I receptors are selectively activated by PACAP, whereas type II receptors bind PACAP and VIP with similar affinity (3). Three isoforms of PACAP receptors have now been cloned and designated as PACAP-specific receptor I (PAC 1 -R) (4, 5) and VIP/PACAP mutual receptors 1 and 2 (VPAC 1 -R and VPAC 2 -R) (6, 7). Both PAC 1 -R (type 1 receptors) and VPAC 1 -R/VPAC 2 -R (type 2 receptors) belong to the seven-transmembrane domain, G-protein-coupled receptor family, and are all positively coupled to adenylyl cyclase (2). Eight isoforms of PAC 1 -R, resulting from alternative splicing, have been characterized to date. These variants display differential signal transduction properties with regard to adenylyl cyclase and phospholipase C (PLC) stimulation (1, 2). In addition to these classical signaling pathways, PACAP has been found to stimulate a Ca 2ϩ -calmodulin nitric oxide synthase (8) and mitogen-activated protein kinase activity (9). These various transduction mechanisms are involved in the neurotrophic activities exerted by PACAP (i.e. inhibition of apoptosis and stimulation of neurite outgrowth) during development (9 -11).PACAP and its receptors are actively expressed in the adrenal medulla (12)(13)(14). In particular, we have previously demonstrated the occurrence of PACAP-38 (15) and PACAP binding sites (16) in chromaffin cells from 16-to 20-week-old fetal human adrenal glands. Activation of these receptors by PACAP-38 causes stimulation of cAMP production and induces a modest increase in inositol 1,4,5-triphosphate (IP 3 ) formation (16), suggesting a role for the neuropeptide in the developing

The aim of the present study was to characterize distribution and pharmacological properties of angiotensin II (Ang II) receptors in human fetal adrenals frozen immediately after removal. Autoradiographic studies indicate that Ang II receptors are present throughout the gland. Coincubations with DUP 753, a specific antagonist of the AT1 receptor, and with PD 123319, a specific antagonist of the AT2 receptor, reveal that Ang II receptors are mainly of the type 2. The AT1 receptors are detected after 16 weeks of gestation at the periphery of the gland. Binding of 125I-Ang II to membrane preparations is dose-dependent and saturable. Competition studies and Scatchard analysis reveal a homogenous population of high-affinity AT2 binding sites (Kd = 0.68 +/- 0.1 nmol/L). Binding capacities decrease from 1080 +/- 304 fmol/mg protein at 14 weeks to 275 +/- 55 fmol/mg protein at 21 weeks. However, when fetal adrenal cells are prepared and cultured for 6 days, the proportion of AT1 receptors increases, indicating that culture conditions induce expression of the AT1 receptor. These results differ from those obtained in adult glands, where autoradiographic studies reveal that the AT1 receptors are found mainly in zona glomerulosa and AT2 receptors mainly in the medulla. These data suggest that the AT2 receptors could be involved in the morphological or functional differentiation of the human fetal adrenal gland.

The distribution and pharmacological properties of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors were studied in the fetal human adrenal gland during the second trimester of gestation. Autoradiographic studies, using [125I]PACAP27 as a radioligand, revealed that PACAP-binding sites are exclusively located on chromaffin cells of adrenals from fetuses 14-20 weeks old. Biochemical characterization of binding revealed the occurrence of a single class of PACAP-binding sites with a dissociation constant value of 0.32-0.74 nmol/L and a binding capacity of 0.30-0.81 pmol/mg wet tissue. PACAP27 and PACAP38 were equipotent in competing for [125I]PACAP27 binding (IC50 = 0.28-0.64 nmol/L and 0.15-0.81 nmol/L, respectively), and the Hill coefficients were close to 1. In contrast, vasoactive intestinal polypeptide was much less efficient in displacing the tracer (IC50 = 4-362 nmol/L), and the Hill coefficients were less than 0.6. PACAP38 induced a dose-dependent increase in cAMP production in fetal human adrenal cell suspension (ED50 = 0.07 +/- 0.02 nmol/L), as well as in cells maintained in culture for 5 days (5.4 +/- 1.8 nmol/L). In contrast, PACAP38 induced a modest increase in inositol phosphate formation. These data indicate that type I PACAP receptors are present in the early stages of the human medulla organization during the process of migration of chromaffin cells from the periphery to the central part of the gland. The present results suggest that PACAP could be involved in the regulation of the human adrenochromaffin cells during ontogenesis.