The role of metallothionein in oncogenesis and cancer prognosis

The antiapoptotic, antioxidant, proliferative, and angiogenic effects of metallothionein (MT)-I+II has resulted in increased focus on their role in oncogenesis, tumor progression, therapy response, and patient prognosis. Studies have reported increased expression of MT-I+II mRNA and protein in various human cancers; such as breast, kidney, lung, nasopharynx, ovary, prostate, salivary gland, testes, urinary bladder, cervical, endometrial, skin carcinoma, melanoma, acute lymphoblastic leukemia (ALL), and pancreatic cancers, where MT-I+II expression is sometimes correlated to higher tumor grade/stage, chemotherapy/radiation resistance, and poor prognosis. However, MT-I+II are downregulated in other types of tumors (e.g. hepatocellular, gastric, colorectal, central nervous system (CNS), and thyroid cancers) where MT-I+II is either inversely correlated or unrelated to mortality. Large discrepancies exist between different tumor types, and no distinct and reliable association exists between MT-I+II expression in tumor tissues and prognosis and therapy resistance. Furthermore, a parallel has been drawn between MT-I+II expression as a potential marker for prognosis, and MT-I+II's role as oncogenic factors, without any direct evidence supporting such a parallel. This review aims at discussing the role of MT-I+II both as a prognostic marker for survival and therapy response, as well as for the hypothesized role of MT-I+II as causal oncogenes.     

Flexibility contra Stiffness: The Phragmoplast as a Physical Barrier for Beads But Not for Vesicles

In plant cells, Golgi vesicles are transported to the division plane to fuse with each other, forming the cell plate, the initial membrane-bordered cell wall separating daughter cells. Vesicles, but not organelles, move through the phragmoplast, which consists of two opposing cylinders of microtubules and actin filaments, interlaced with endoplasmic reticulum membrane. To study physical aspects of this transport/inhibition process, we microinjected fluorescent synthetic 1,2-dioleoyl-sn-glycero-3-phospho-rac-1-glycerol (DOPG) vesicles and polystyrene beads into Tradescantia virginiana stamen hair cells. The phragmoplast was nonselective for DOPG vesicles of a size up to 150 nm in diameter but was a physical barrier for polystyrene beads having a diameter of 20 and 40 nm and also when beads were coated with the same DOPG membrane. We conclude that stiffness is a parameter for vesicle transit through the phragmoplast and discuss that cytoskeleton configurations can physically block such transit.Cells and their constituents are physical entities, and next to chemical interactions, cell structures are determinants of cell behavior. Therefore, apart from techniques to image living cells at the subcellular level, experiments are needed that probe physical parameters important in cell function in vivo. We took the plant phragmoplast structure to answer the question whether the physical aspect “stiffness” is a factor in the inhibition of transport through this structure by microinjecting synthetic vesicles and polystyrene beads in Tradescantia virginiana stamen hair cells during cytokinesis, when the phragmoplast is essential for partitioning the cytoplasm between two daughter cells. Plant cells partition by producing a cell plate made of fused 60- to 80-nm-diameter vesicles (Staehelin and Hepler, 1996; Jürgens, 2005) proven to be Golgi vesicles (Reichardt et al., 2007). Their content becomes the new cell wall and their membranes become the daughter cell plasma membranes. The phragmoplast consists of two opposing cylinders of microtubules and actin filaments, interlaced with similarly aligned endoplasmic reticulum (ER) membranes. This phragmoplast cytoskeleton is the transport vehicle for Golgi vesicles to the plane where the cell plate is being formed (Staehelin and Hepler, 1996; Valster et al., 1997), keeps them in this plane (Esseling-Ozdoba et al., 2008b), where they fuse with each other (Samuels et al., 1995; Otegui et al., 2001; Seguí-Simarro et al., 2004), and assists in the proper attachment of the cell plate to the parental cell wall (Valster et al., 1997; Molchan et al., 2002). Transit of organelles, including Golgi bodies, is inhibited (Staehelin and Hepler, 1996; Nebenführ et al., 2000; Seguí-Simarro et al., 2004). Most of these data are known from static electron microscopy images. Electron microscopy after high-pressure freezing and freeze substitution (Thijsen et al., 1998) and electron tomography studies (Otegui et al., 2001; Seguí-Simarro et al., 2004; Austin et al., 2005) show that, in the early stage of cell plate formation in the center and later at the phragmoplast border, microtubules are aligned parallel to each other at distances of 20 to 100 nm. Keeping in mind that also actin filaments and ER membranes, aligned in the same orientation, are present between the microtubules, this leaves little room for the cell plate-forming vesicles during their transport through this phragmoplast.Clearly, during the past decade, significant progress has been made in the elucidation of the structural organization of cell plate-forming phragmoplasts, which has set the stage for studies to elucidate physical properties of phragmoplasts. The experimental approach we use is injecting particulate and vesicular fluorescent probes into living and dividing cells and observing the extent to which such probes can enter the phragmoplast and can be transported to the cell plate region. We have shown before that synthetic lipid 1,2-dioleoyl-sn-glycero-3-phospho-rac-1-glycerol (DOPG) vesicles of 60 nm diameter are transported through the phragmoplast, accumulate, and are kept in the cell plate region but do not fuse (Esseling-Ozdoba et al., 2008b). Now, we asked whether similar, flexible, synthetic lipid (DOPG) vesicles of various sizes, smaller and larger than endogenous vesicles, as well as stiff polystyrene beads, and such beads coated with the DOPG membrane, are transported through the phragmoplast and enter the plane where the cell plate is being formed, a question pertaining to a physical property of the phragmoplast. Our principal finding is that injected synthetic vesicles up to 150 nm diameter can enter and be transported to the cell plate region, where they accumulate but do not become incorporated into the cell plate. In contrast, polystyrene beads, the noncoated ones and those coated with the same lipid as the vesicles with diameters of 20 and 40 nm, can enter phragmoplasts but cannot be transported to the cell plate region, and the 40-nm beads slow cell plate formation, possibly by interfering with the delivery of normal, cell plate-forming vesicles to the cell plate.     

Differential functions of genes regulated by VEGF-NFATc1 signaling pathway in the migration of pulmonary valve endothelial cells

We have reported that vascular endothelial growth factor (VEGF)-A induces the proliferation of human pulmonary valve endothelial cells (HPVECs) through nuclear factor in activated T cells (NFAT)c1 activation [1]. Here we show that VEGF-A increases the migration of HPVECs through NFATc1 activation, suggesting that VEGF-A/NFATc1 regulates the migration of HPVECs. To learn how this pathway may be involved in post-natal valvular repair, HPVECs were treated with VEGF-A, with or without cyclosporine A to selectively block VEGF-NFATc1 signaling. Down Syndrome critical region 1 (DSCR1) and heparin-binding EGF-like growth factor (HB-EGF) are two genes identified by DNA microarray as being up-regulated by VEGF-A in a cyclosporine-A-sensitive manner. DSCR1 silencing increased the migration of ovine valve endothelial cells, whereas HB-EGF silencing inhibited migration. This differential effect suggests that VEGF-A/NFATc1 signaling might be a crucial coordinator of endothelial cell migration in post-natal valves.     

Heterologous expression of the AtDREB1A gene in chrysanthemum increases drought and salt stress tolerance

DNA cassette containing an AtDREB1A cDNA and a nos terminator, driven by a cauliflower mosaic 35S promoter, or a stress-inducible rd29A promoter, was transformed into the ground cover chrysanthemum (Dendranthema grandiflorum) ‘Fall Color’ genome. Compared with wild type plants, severe growth retardation was observed in 35S:DREB1A plants, but not in rd29A:DREB1A plants. RT-PCR analysis revealed that, under stress conditions, the DREB1A gene was over-expressed constitutively in 35S:DREB1A plants, but was over-expressed inductively in rd29A:DREB1A plants. The transgenic plants exhibited tolerance to drought and salt stress, and the tolerance was significantly stronger in rd29A:DREB1A plants than in 35S:DREB1A plants. Proline content and SOD activity were increased inductively in rd29A:DREB1A plants than in 35S:DREB1A plants under stress conditions. These results indicate that heterologous AtDREB1A can confer drought and salt tolerance in transgenic chrysanthemum, and improvement of the stress tolerance may be related to enhancement of proline content and SOD activity.     

Heterologous expression of the AtDREB1A gene in chrysanthemum increases drought and salt stress tolerance

DNA cassette containing an AtDREB1A cDNA and a nos terminator,driven by a cauli- flower mosaic 35S promoter,or a stress-inducible rd29A promoter,was transformed into the ground cover chrysanthemum(Dendranthema grandiflorum)'Fall Color'genome.Compared with wild type plants,severe growth retardation was observed in 35S:DREB1A plants,but not in rd29A:DREB1A plants.RT-PCR analysis revealed that,under stress conditions,the DREB1A gene was over-expressed constitutively in 35S:DREB1A plants,but was over-expressed inductively in rd29A:DREB1A plants.The transgenic plants exhibited tolerance to drought and salt stress,and the tolerance was significantly stronger in rd29A:DREB1A plants than in 35S:DREB1A plants.Proline content and SOD activity were increased inductively in rd29A:DREB1A plants than in 35S:DREB1A plants under stress conditions.These results indicate that heterologous AtDREB1A can confer drought and salt tolerance in transgenic chrysanthemum,and improvement of the stress tolerance may be related to enhancement of proline content and SOD activity.     

Formation of the marginal "pro-oxidant" zone and its role in the enhancement of lipid peroxidation in the area of myocardial ischemia and infarction

The formation of peripheral zone devoid of dehydrogenase activity but possessing vessels connected with the normal myocardium was demonstrated in the area of fresh myocardial infarction 2 h after coronary occlusion. A direct correlation between the changes of the zone area and the intensity of free radical lipid peroxidation in the area of fresh myocardial infarction was established.     

3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-Δ⁸-THC analogues, several of which have high affinity for the CB₂ receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-Δ⁸-THC (5), 1-deoxy-Δ⁸-THC (6), 1-deoxy-3-butyl-Δ⁸-THC (7), 1-deoxy-3-hexyl-Δ⁸-THC (8) and a series of 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=0–4, 6, 7, where n=the number of carbon atoms in the side chain−2). Three derivatives (17–19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB₁ and CB₂ receptors were determined employing previously described procedures. Five of the 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=1–5) have high affinity (K_i=<20 nM) for the CB₂ receptor. Four of them (2, n=1–4) also have little affinity for the CB₁ receptor (K_i=>295 nM). 3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ⁸-THC (2, n=2) has very high affinity for the CB₂ receptor (K_i=3.4±1.0 nM) and little affinity for the CB₁ receptor (K_i=677±132 nM).

Scheme 3. (a) (C₆H₅)₃PCH₃⁺ Br⁻, n-BuLi/THF, 65°C; (b) LiAlH₄/THF, 25°C; (c) KBH(sec-Bu)₃/THF, −78 to 25°C then H₂O₂/NaOH.     

Transgenic mice expressing osteopontin in hepatocytes as a model of autoimmune hepatitis

Osteopontin, a crucial factor for Th1 immune response, is expressed in stellate cells and macrophages activated in injured liver. To clarify the role of osteopontin in inflammatory changes in the liver, we attempted to establish transgenic mice expressing osteopontin in hepatocytes. Mouse osteopontin cDNA, cloned from concanavalin-A-stimulated spleen cells in C57BL/6 mice, was constructed into the vector containing serum amyloid-P component promoter. This construction was microinjected into fertilized eggs of C57BL/6 mice, and 4 lines of the transgenic mice were obtained. Western blotting and immunohistochemistry revealed that osteopontin was expressed in hepatocytes, but not in non-parenchymal cells, in the transgenic mice. The mean osteopontin concentrations in the liver and plasma in the mice were 13 and 2.6 times higher than those in negative littermates. Antinuclear antibody was positive in the plasma in 50% of the transgenic mice. In the transgenic mice later than 12 weeks of age, mononuclear cell infiltration in the liver developed, and these cells were positive for CD8 and HLA-DR. Plasma ALT activity was increased with focal necrosis in hepatic lobules in the transgenic mice later than 24 weeks of age. The transgenic mice expressing osteopontin in hepatocytes may be useful as a model of autoimmune hepatitis.     

Genetic polymorphims in promoter region of osteopontin gene may be a marker reflecting hepatitis activity in chronic hepatitis C patients

BACKGROUND AND AIMS: Osteopontin, an extracellular matrix protein with RGD motif, is shown to be a cytokine essential for Th1 immune response initiation. Genetic polymorphisms in the osteopontin gene (OPN) determine the magnitude of immunity against rickettsial infection in mice. Similar polymorphisms, if present also in human beings, might affect hepatitis activity in those infected with HCV. METHODS: Blood was collected from 176 patients with chronic hepatitis C. SNPs in the promoter region of OPN were analyzed in 20 patients by direct sequencing of DNA fragments amplified by PCR and in 156 patients by Invader assay. Ninety-five patients compatible to evaluation criteria were classified into three groups depending on maximal serum ALT levels during the observation periods at least for 2 years as follows; lower than 30IU/L (low-activity group), between 30 and 80IU/L with no hepatoprotective treatment (medium-activity group), and higher than 80IU/L irrespective of hepatoprotective treatment (high-activity group). RESULTS: There were 16, 19, and 60 patients in the low-, medium-, and high-activity groups, respectively. Four SNPs (nt -155, -443, -616, and -1748) were detected in the promoter region of OPN. Among them, the SNP at nt -443 (C or T) was a novel one and showed an association with hepatitis activity in our patients: T/T homozygosity was found in 2 (13%), 8 (42%), and 25 (44%), and C/T heterozygosity in 12 (75%), 8 (42%), and 23 (40%), in the low-, medium-, and high-activity groups, respectively. The other 3 SNPs already known showed linkage disequilibrium with D(') and r(2) greater than 0.937 to each other without correlation to disease activity. CONCLUSIONS. OPN promoter region SNP at nt -433 may be a useful marker reflecting hepatitis activity in chronic hepatitis C patients.