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The ErbB2 (Her2/neu epidermal growth receptor family) oncogene is overexpressed in 30% to 40% of human breast cancers. Cyclin D1 is the regulatory subunit of the holoenzyme that phosphorylates and inactivates the retinoblastoma (pRb) tumor suppressor and is an essential downstream target of ErbB2-induced tumor growth. Herein, we demonstrate that ErbB2 induces the activity of the Notch signaling pathway. ErbB2 induction of DNA synthesis, contact-independent growth, and mammosphere induction required Notch1. ErbB2-induced cyclin D1 and cyclin D1 expression was suficient to induce Notch1 activity, and conversely, genetic deletion of Notch1 in mammary epithelial cells using foxed Notch (Notch(fl/fl)) mice demonstrated that cyclin D1 is induced by Notch1. Genetic deletion of cyclin D1 or small interfering RNA (siRNA) to cyclin D1-reduced Notch1 activity and reintroduction of cyclin D1 into cyclin D1-deficient cells restored Notch1 activity through the inhibition of Numb, an endogenous inhibitor of Notch1 activity. Thus, cyclin D1 functions downstream as a genetic target of Notch1, amplifies Notch1 activity by repressing Numb, and identifies a novel pathway by which ErbB2 induces Notch1 activity via the induction of cyclin D1.

Tumor necrosis factor (TNF) family member, the receptor activator of NF-κB ligand (RANKL) is expressed on marrow stromal/osteoblast cells in response to several osteotropic factors and is critical for osteoclast precursor differentiation to form multinucleated osteoclasts, which resorb bone [Lacey et al., 1998; Hsu et al., 1999; Kong et al., 1999]. Enhanced levels of RANKL are associated with pathologic conditions such as Paget's disease of bone [Menaa et al., 2000; Neale et al., 2000] and multiple myeloma [Roux et al., 2002]. Several osteotropic factors such as l,25-(OH)2D3, parathyroid hormone (PTH), interleukin 1β (IL-1β), interleukin-11, and prostaglandin E2 (PGE2) induce osteoclast differentiation through enhanced expression of RANKL in marrow stromal/osteoblast cells [Nakashima et al., 2000; Lee et al., 2002], but the molecular mechanisms which regulate RANKL gene expression are unclear. It has been reported that IL-1β and TNF-α stimulate RANKL expression in human bone marrow stromal cells through activation of p38 MAP kinase pathway [Rossa et al., 2006]. In addition, fibroblast growth factor-2 (FGF-2) has been shown to induce RANKL production through COX-2-mediated prostaglandin synthesis and by suppressing osteoclastogenesis inhibitory factor in mouse osteoblastic cells [Nakagawa et al., 1999]. Similarly, lipopolysaccharide treatment increased the levels of RANKL expression through activation of Toll-like receptors in primary murine osteoblast cells [Kikuchi et al., 2001]. Furthermore, transforming growth factor β (TGF-β) has been shown to increase RANKL expression in activated T-cells by increasing anti-CD3 [Wang et al., 2002]. Recently, it has also been reported that PTH stimulates RANKL expression through cAMP/protein kinase A/CREB cascade [Fu et al., 2006]. We recently demonstrated that heat shock factor-2 (HSF-2) is a downstream target of FGF-2 signaling to induce RANKL expression in bone marrow stromal/preosteoblast cells [Roccisana et al., 2004]. Heat shock proteins (HSP) are molecular chaperones expressed in cells in response to a variety of stimuli such as temperature and stimulation of membrane-bound receptors by hormones/cytokines and other chemical factors. Thus, HSP are an integral part of mammalian development [Christians et al., 2003]. Heat shock transcription factors (HSF), which binds to the heat shock responsive element (HSE), modulate expression of HSP and several other genes including TNF-α family [Mathew et al., 1998; Snoeckx et al., 2001]. Although multiple osteotropic factors including FGF-2 are known to modulate RANKL gene expression in the bone microenvironment, the transcriptional regulatory mechanisms operative in marrow stromal/preosteoblast cells are not well established. DACH1 is a human homolog of the Drosophila dachshund gene, which is a key regulator for organ development and is considered a cell fate determination factor. DACH1 contains a conserved domain (dachshund domain (DS)) in the N-terminal region that is structurally homologous with the Ski and Sno proto-oncogenes and interacts with the nuclear co-repressor NCoR to modulate transcription factor activity. We have previously reported that DACH1 represses TGF-β signaling through binding to Smad4 [Wu et al., 2003]. Two vertebrate homologues, Dach1 and Dach2 are partially functionally redundant, since Dach1−/− mice survive to birth but exhibit postnatal lethality associated with a failure to suckle, cyanosis, and respiratory distress [Davis et al., 2001]. DACH1 expression is lost in a subset of human breast cancers associated with poor prognosis. Reintroduction of DACH1 inhibits breast tumor cell proliferation and tumor growth through suppression of cyclin D1 [Wu et al., 2006]. DACH1 inhibits TGF-β signaling in ovarian cancer cells [Sunde et al., 2006]. These studies are part of a growing body of evidence that DACH1 may function as a tumor suppressor. Recent evidence indicates that DACH1 is associated with FGF signaling during limb and skeletal development and may regulate cell proliferation or differentiation [Horner et al., 2002]. Dach1 binds with Runx2/Cbfa1 and co-localizes with cyclin-dependent kinase inhibitor p27 (Kip1) and p57 (Kip2) at the growth plate region in chondrocytes [Horner et al., 2002]. In this study, we show that DACH1 negatively regulate RANKL gene expression in human bone marrow-derived stromal/preosteoblast cells and that FGF-2 signaling promotes HSF-2 binding to DACH1 to modulate RANKL gene expression in these cells.

The position effect variegation in Drosophila and Schizosaccharomyces pombe, and higher-order chromatin structure regulation in yeast, is orchestrated by modifier genes of the Su(var) group, (e.g., histone deacetylases ([HDACs]), protein phosphatases) and enhancer E(Var) group (e.g., ATP [adenosine 5'-triphosphate]-dependent nucleosome remodeling proteins). Higher-order chromatin structure is regulated in part by covalent modification of the N-terminal histone tails of chromatin, and histone tails in turn serve as platforms for recruitment of signaling modules that include nonhistone proteins such as heterochromatin protein (HP1) and NuRD. Because the enzymes governing chromatin structure through covalent modifications of histones (acetylation, methylation, phosphorylation, ubiquitination) can also target nonhistone substrates, a mechanism is in place by which epigenetic regulatory processes can affect the function of these alternate substrates. The posttranslational modification of histones, through phosphorylation and acetylation at specific residues, alters chromatin structure in an orchestrated manner in response to specific signals and is considered the basis of a "histone code." In an analogous manner, specific residues within transcription factors form a signaling module within the transcription factor to determine genetic target specificity and cellular fate. The architecture of these signaling cascades in transcription factors (SCITs) are poorly understood. The regulation of estrogen receptor (ERalpha) by enzymes that convey epigenetic signals is carefully orchestrated and is reviewed here.

The BRCA1 gene was identified and cloned in 1994 based its linkage to early onset breast cancer and breast-ovarian cancer syndromes in women. While inherited mutations of BRCA1 are responsible for about 40-45% of hereditary breast cancers, these mutations account for only 2-3% of all breast cancers, since the BRCA1 gene is rarely mutated in sporadic breast cancers. However, BRCA1 expression is frequently reduced or absent in sporadic cancers, suggesting a much wider role in mammary carcinogenesis. Since BRCA1 was cloned in 1994, its molecular function has been the subject of intense investigation. These studies have revealed multiple functions of the BRCA1 that may contribute to its tumor suppressor activity, including roles in: cell cycle progression, several highly specialized DNA repair processes, DNA damage-responsive cell cycle check-points, regulation of a set of specific transcriptional pathways, and apoptosis. Many of these functions are linked to protein:protein interactions involving different portions of the 1,863 amino acid (aa) BRCA1 protein. BRCA1 functions in cell cycle progression and the DNA damage response appear to be regulated by distinct and specific phosphorylation events, but the molecular pathways activated by these phosphorylations are only beginning to be unraveled. In addition, the reason that BRCA1 mutation carriers develop specific tumor types (breast and ovarian cancers in women and possibly prostate cancers in men) is not clearly understood. Elucidation of the precise molecular functions of the BRCA1 gene product will greatly enhance our understanding of the pathogenesis of hereditary as well as sporadic mammary carcinogenesis.

Microglia, the resident brain macrophages, are the principal cells involved in the regulation of inflammatory and antimicrobial responses in the CNS. Interferon-beta (IFNbeta) is an antiviral cytokine induced by viral infection or following non-specific inflammatory challenges of the CNS. Because of the well-known anti-inflammatory properties of IFNbeta, it is also used to treat multiple sclerosis, an inflammatory CNS disease. Despite the importance of IFNbeta signaling in CNS cells, little has been studied, particularly in microglia. In this report, we investigated the molecular mechanisms underlying IFNbeta-induced beta-chemokine expression in primary human fetal microglia. Multiple signaling cascades are activated in microglia by IFNbeta, including nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and Jak/Stat. IFNbeta induced IkappaBalpha degradation and NF-kappaB (p65:p50) DNA binding. Inhibition of NF-kappaB by either adenoviral transduction of a super repressor IkappaBalpha, or an antioxidant inhibitor of NF-kappaB reduced expression of the beta-chemokines, regulated upon activation, normal T-cell expressed and secreted (RANTES) and macrophage inflammatory protein (MIP)-1beta. IFNbeta also induced phosphorylation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase, and the MAP kinase kinase 1 (MEK1) inhibitor PD98059 dose-dependently inhibited beta-chemokine mRNA and protein expression. PD98059 did not inhibit NF-kappaB binding, demonstrating that ERK was not responsible for NF-kappaB activation. Two downstream targets of ERK were identified in microglia: AP-1 and Stat1. IFNbeta induced AP-1 nuclear binding activity in microglia and this was suppressed by PD98059. Additionally, IFNbeta induced Stat1 phosphorylation at both tyrosine 701 (Y701) and serine 727 (S727) residues. S727 phosphorylation of Stat1, which is known to be required for maximal transcriptional activation, was inhibited by PD98059. Our results demonstrating multiple signaling cascades initiated by IFNbeta in primary human microglia are novel and have implications for inflammatory and infectious diseases of the CNS.

Background The applications of traditional retroviral vectors are limited because proviral integrations into the host genome require DNA synthesis. Lentiviruses are considered to be advantageous because of their ability to infect non-dividing cells. Methods To demonstrate the potential of lentiviral vectors, we used a human immunodeficiency virus (HIV)-1 virus encoding the green fluorescence protein (GFP) to infect fetal human hepatocytes. GFP-expressing cells were transplanted into the liver of Balb/C SCID mice via intrasplenic injection. Results Primary fetal hepatocytes incorporated the GFP reporter with high (30–40%) efficiency. A cell line derived from human fetal liver (HFL) exhibited similar transduction efficiency to the lentiviral vector. To demonstrate the relationship between lentiviral gene transfer and cell proliferation, cells were subjected to gamma-irradiation, which attenuated the replication of primary fetal hepatocytes. However, lentiviral gene transfer was unaffected by this decrease in cell proliferation. GFP expression in transduced cells was preserved during multiple passages in cell culture. When GFP-expressing cells were transplanted into the liver of Balb/C SCID mice via intrasplenic injection, GFP expression was observed throughout the 3 week duration of the study. Conclusion These studies establish that human hepatocytes are amenable to lentiviral gene transfer with sustained transgene expression. Incorporation of lentiviral vectors will be helpful in testing strategies for hepatic gene therapy. Copyright © 2000 John Wiley & Sons, Ltd.

The Tau-1 monoclonal antibody was localized to the nucleolus of interphase cells and the nucleolar organizing regions (NORs) of acrocentric chromosomes in cultured human cells. Putative nucleolar and NOR tau was found in CG neuroblastoma cells which contain nucleolar tau and little cytoplasmic tau. To further establish the presence of tau in the nucleolus of these cells, sense and anti-sense transfection strategies were used. CG neuroblastoma cells were transfected with tau sense cDNA and immunostained with Tau-1. Cytoplasmic Tau-1 staining was greatly increased in CG cells which contain very little endogenous cytoplasmic tau. Nucleolar Tau-1 staining was also increased in certain CG cells indicating an increase in nucleolar tau in a subset of transfected cells. CG cells were also transfected with tau anti-sense cDNA which abolished Tau-1 staining in the nucleolus. These results contribute to a growing body of evidence defining tau as a multifunctional protein found in both the cytoplasm and nucleoli of primate cells.

1. To examine individual hormonal responses to extreme physical stress, blood samples were taken from eight highly trained athletes 1 day before and within 15 min of finishing the 1986 1000 km Sydney to Melbourne Ultramarathon foot race. 2. The baseline hormonal state of these highly trained athletes was quite different from normal. Resting serum conjugated catecholamines--epinephrine (E), norepinephrine (NE), dopamine (D), free E and free D--were significantly elevated above the normal mean (P less than 0.01). ACTH levels were significantly elevated above the normal range. Immunoreactive beta-endorphin (IR-beta EP), growth hormone (GH), prolactin (PRL), testosterone, cortisol and cortisol-binding globulin (CBG) were within the normal range. 3. The effect of the race on serum catecholamine levels was to elevate further free and conjugated NE (P less than 0.01). Other catecholamines, free and conjugated, remained significantly elevated above the normal mean (P less than 0.01). Adrenocorticotrophic hormone (ACTH) remained elevated, and IR-beta EP within the normal range, without significant change. A significant increase in GH (P less than 0.05), PRL (P less than 0.01), and cortisol (P less than 0.01) was seen, with no change in CBG. 4. As a model of chronic physical stress, the ultramarathon runner demonstrates a significantly altered baseline hormonal state as reflected in the primary mediators of the stress response, the catecholamines and the hypothalamic-pituitary-adrenal axis. Their response to severe exercise is distinct from that of untrained individuals in whom conjugated catecholamines decrease and ACTH increase. This may represent hormonal adaptation to prolonged stress.