Constitutive tyrosine phosphorylation of ErbB-2 via Jak2 by autocrine secretion of prolactin in human breast cancer

Overexpression of the oncogene for ErbB-2 is an unfavorable prognostic marker in human breast cancer. Its oncogenic potential appears to depend on the state of tyrosine phosphorylation. However, the mechanisms by which ErbB-2 is constitutively tyrosine-phosphorylated in human breast cancer are poorly understood. We now show that human breast carcinoma samples with ErbB-2 overexpression have higher proliferative and metastatic activity in the presence of autocrine secretion of prolactin (PRL). By using a neutralizing antibody or dominant negative (DN) strategies or specific inhibitors, we also show that activation of Janus kinase Jak2 by autocrine secretion of PRL is one of the significant components of constitutive tyrosine phosphorylation of ErbB-2, its association with Grb2 and activation of mitogen-activated protein (MAP) kinase in human breast cancer cell lines that overexpress ErbB-2. Furthermore, the neutralizing anti-PRL antibody or erbB-2 antisense oligonucleotide or DN Jak2 or Jak2 inhibitor or DNRas or MAP kinase kinase inhibitor inhibits the proliferation of both untreated and PRL-treated cells. Our results indicate that autocrine secretion of PRL stimulates tyrosine phosphorylation of ErbB-2 by Jak2, provides docking sites for Grb2 and stimulates Ras-MAP kinase cascade, thereby causing unrestricted cellular proliferation. The identification of this novel cross-talk between ErbB-2 and the autocrine growth stimulatory loop for PRL may provide new targets for therapeutic and preventive intervention of human breast cancer.     

Reciprocal role of ERK and NF-kappaB pathways in survival and activation of osteoclasts

To examine the role of mitogen-activated protein kinase and nuclear factor kappa B (NF-kappaB) pathways on osteoclast survival and activation, we constructed adenovirus vectors carrying various mutants of signaling molecules: dominant negative Ras (Ras(DN)), constitutively active MEK1 (MEK(CA)), dominant negative IkappaB kinase 2 (IKK(DN)), and constitutively active IKK2 (IKK(CA)). Inhibiting ERK activity by Ras(DN) overexpression rapidly induced the apoptosis of osteoclast-like cells (OCLs) formed in vitro, whereas ERK activation after the introduction of MEK(CA) remarkably lengthened their survival by preventing spontaneous apoptosis. Neither inhibition nor activation of ERK affected the bone-resorbing activity of OCLs. Inhibition of NF-kappaB pathway with IKK(DN) virus suppressed the pit-forming activity of OCLs and NF-kappaB activation by IKK(CA) expression upregulated it without affecting their survival. Interleukin 1alpha (IL-1alpha) strongly induced ERK activation as well as NF-kappaB activation. Ras(DN) virus partially inhibited ERK activation, and OCL survival promoted by IL-1alpha. Inhibiting NF-kappaB activation by IKK(DN) virus significantly suppressed the pit-forming activity enhanced by IL-1alpha. These results indicate that ERK and NF-kappaB regulate different aspects of osteoclast activation: ERK is responsible for osteoclast survival, whereas NF-kappaB regulates osteoclast activation for bone resorption.     

Cyclin E as a coactivator of the androgen receptor

Androgens play an important role in the growth of prostate cancer, but the molecular mechanism that underlies development of resistance to antiandrogen therapy remains unknown. Cyclin E has now been shown to increase the transactivation activity of the human androgen receptor (AR) in the presence of its ligand dihydrotestosterone. The enhancement of AR activity by cyclin E was resistant to inhibition by the antiandrogen 5-hydroxyflutamide. Cyclin E was shown to bind directly to the COOH terminus portion of the AB domain of the AR, and to enhance its AF-1 transactivation function. These results suggest that cyclin E functions as a coactivator of the AR, and that aberrant expression of cyclin E in tumors may contribute to persistent activation of AR function, even during androgen ablation therapy.     

Orally active cephalosporins. Part 2: synthesis, structure-activity relationships and oral absorption of cephalosporins having a C-3 pyridyl side chain

A series of 7beta-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamid o]cephalosporins having a pyridine ring connected through various spacer moieties at the C-3 position was designed and synthesized and evaluated for antibacterial activity and oral absorption in rats. All compounds showed potent antibacterial activity against Staphylococcus aureus, whereas antibacterial activity against gram-negative bacteria was markedly influenced by the spacer moiety between the pyridine and cephem nucleus. Oral absorption was influenced by the position of the pyridine nitrogen as well as by the spacer moiety. Among these compounds, FR86830 (14), having a 4-pyridylmethylthio moiety at the C-3 position, showed the most well balanced activity and moderate oral absorption.     

High expression of glycogen-debranching enzyme in Escherichia coli and its competent purification method

Glycogen-debranching enzyme (GDE) gene from Saccharomyces cerevisiae was cloned and expressed into Escherichia coli. A 99.3% homology was found between the nucleotide sequences of GDE gene harbored in the recombinant E. coli plasmid (pTrc99A) and the open reading frame (902039-906646 position) of the 4608-bp fragment of S. cerevisiae chromosome XVI. We investigated the best conditions for GDE expression. When the cultivation temperature of recombinant E. coli strains was lowered to 25 degrees C and the isopropyl-beta-d-thiogalactopyranoside (IPTG) concentration used for induction was decreased to as low as 0.02 mM, a total of about 33 mg of recombinant GDE can be isolated from a liter culture as estimated by amylo-1,6-glucosidase activity. Consecutively, we developed a new method for purifying GDE. The method requires only a single-step purification via beta-cyclodextrin-immobilized Sepharose 6B (beta-CD Sepharose 6B) affinity chromatography and renders a 90% recovery of the enzyme. Moreover, the purified recombinant GDE is a homogeneous protein and possesses the same characteristics as those of S. cerevisiae. With the highly expressed GDE in recombinant E. coli and a rapid and effective purification method, we successfully resolved the hurdle always faced for obtaining an ample amount of purified GDE. The availability of GDE, hence, may allow advancement on GDE studies and provide new prospects for GDE on biotechnological application.     

Mechanism of alpha-tocopheryl succinate-induced apoptosis of promyelocytic leukemia cells

Selective induction of apoptosis in tumor cells is important for treating patients with cancer. Because oxidative stress plays an important role in the process of apoptosis, we studied the effect of alpha-tocopheryl succinate (VES) on the fate of cultured human promyelocytic leukemia cells (HL-60). The presence of fairly low concentrations of VES inhibited the growth and DNA synthesis of HL-60 cells, and also induced their apoptosis via a mechanism that was inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), an inhibitor of pan-caspases. VES activated various types of caspases, including caspase-3, 6, 8, and 9, but not caspase-1. VES triggered the reaction leading to the cleavage of Bid, a member of the death agonist Bcl-2 family, and released cytochrome c (Cyt.c) from the mitochondria into the cytosol by a z-VAD-fmk-inhibitable mechanism. VES transiently increased the intracellular calcium level [Ca2+]i and stimulated the release of Cyt.c in the presence of inorganic phosphate (Pi). However, high concentrations of VES (approximately 100 microM) hardly induced swelling of isolated mitochondria but depolarized the mitochondrial membrane potential by a cyclosporin A (CsA)-insensitive mechanism. These results indicate that VES-induced apoptosis of HL-60 cells might be caused by activation of the caspase cascade coupled with modulation of mitochondrial membrane function.     

Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts

For introducing regions of human chromosomes greater than a megabase into cells or animals, we have developed a chromosome-cloning system in which defined regions of human chromosomes can be cloned into a stable human minichromosome vector in homologous recombination-proficient chicken DT40 cells. The stable minichromosome vector allowed a 10 Mb-sized region of the mitotically unstable human chromosome 22 to be stably maintained in mouse embryonic stem (ES) cells, and in mice. Furthermore, we demonstrated functional expression of human genes from the HAC in mice. This study describes a stable cloning and expression system for greater than megabase-sized regions of human chromosomes.     

Pigment cell-specific expression of the tyrosinase gene in ascidians has a different regulatory mechanism from vertebrates

Tyrosinase is the key enzyme required for the synthesis of melanin pigments. Sequence comparison and functional analysis of the 5' upstream regions of vertebrate tyrosinase genes have revealed the importance of conserved E-box motifs in regulating their specific expression in pigment cells, optic cup-derived retinal pigment epithelium (RPE) and neural crest-derived melanocytes. In ascidians (more basal protochordates), two pigment cells that resemble vertebrate RPE cells are formed and specifically express the orthologous tyrosinase gene (HrTyr) in the cerebral vesicle located at the anterior end of the neural tube. To define regulatory sequences required for pigment cell-lineage-specific expression of HrTyr during embryogenesis, a series of mutations of the 5' upstream region of HrTyr were fused to the lacZ reporter gene and were microinjected into fertilized eggs. We found that the -152bp upstream of the translational start site is essential for expression in pigment cell precursors of tailbud-stage embryos. Further, additional positive and unique restriction elements were identified in the region up to -1.8kb. Surprisingly, in the -152bp minimal promoter or in other regions with regulatory activities, there are no E-box motifs or sequences correlating with other conserved elements regulating vertebrate tyrosinase promoters. The possibility that Pax proteins regulate HrTyr expression is also discussed.     

Survey on antibody against parapoxvirus among cattle in Japan

A seroepidemiological survey was performed on antibody against parapoxvirus among cattle in Japan using the agar gel immunodiffusion test and enzyme-linked immunosorbent assay. A total 1,819 sera were collected from cattle in various parts of Japan for the survey. The positive rates were in the range of 40 to 98%, and the reactors increased gradually in number with advancing age. These results indicate that parapoxvirus infection is already prevalent among cattle in Japan. It remains to be elucidated, however, whether the antibodies detected have been produced by infection of the same virus or other viruses that belong to the genus parapoxvirus.     

cDNA and deduced amino acid sequences of apolipophorin-IIIs from Bombyx mori and Bombyx mandarina

The cDNA sequence for apolipophorin-III from two strains of Bombyx mori (N4 and P50) and the Japanese and Chinese strains of Bombyx mandarina were determined. Both the cDNA and deduced amino acid sequences of the four apolipophorin-IIIs were highly similar (95-98%). The four Bombyx sequences also showed significant similarity to the sequence of apolipophorin-III from another lepidopteran, Manduca sexta (83-84%), particularly in the five amphipathic alpha-helices that are proposed to play a critical role in the binding of apolipophorin-III to lipophorin. In the coding region, the nucleotide sequences for the Chinese strain of B. mandarina and the P50 strain of B. mori were identical, supporting the suggestion that P50 is the current strain most closely related to the original domesticated strain. The N4 strain of B. mori is more closely related to these two strains than is the Japanese strain of B. mandarina, suggesting that Japanese strain of B. mandarina separated from the Chinese strain of B. mandarina before domestication of B. mori. Arch.