Role and regulation of c-KIT gene expression in tumor growth and metastasis of human melanoma

The purpose of this study was to investigate the role of the c-KIT receptor in the progression of human melanoma and the mechanism(s) for the regulation of c-KIT gene expression in human melanoma.^ The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP) (metastatic phenotype) are not well-defined. Expression of the tyrosine-kinase receptor c-KIT progressively decreases during local tumor growth and invasion of human melanomas. To provide direct evidence that the metastasis of human melanoma is associated with the loss of c-KIT expression, highly metastatic A375SM cells, which express very low or undetectable levels of c-KIT, were tranduced with the human c-KIT gene. We demonstrated that enforced c-KIT expression in highly metastatic human melanoma cells significantly suppressed their tumorigenicity and metastatic propensity in nude mice. In addition, we showed that the ligand for c-KIT, SCF, induces apoptosis in human melanoma cells expressing c-KIT under both in vitro and in vivo conditions. These results suggest that loss of c-KIT receptor may allow malignant melanoma cells to escape SCF/c-KIT-mediated apoptosis, thus contributing to tumor growth and eventually metastasis.^ Furthermore, we investigated the possible mechanism(s) for the down-regulation of c-KIT gene expression in malignant melanoma. Sequence analysis of the c-KIT promoter indicated that this promoter contains several consensus binding-site sequences including three putative AP2 and two Myb sites. Although Myb was shown to be associated with c-KIT expression in human hemotopoietic cells, we found no correlation between c-KIT expression and Myb expression in human melanoma cell lines. In contrast, we showed that c-KIT expression directly correlates with expression of AP2 in human melanoma cells. We found that highly metastatic cells do not express the transcription factor AP2. Expression of AP2 in A375SM cells (c-KIT-negative and AP2-negative) was enough to restore luciferase activity driven by the c-KIT promoter in a dose-dependent manner. On the other hand, co-expression of the dominant-negative form of AP2 (AP2B) in Mel-501 cells (c-KIT-positive and AP2-positive) resulted in two-fold reduction in luciferase activity. Electrophoretic mobility shift assays revealed that the c-KIT promoter contains functional AP2 binding sites which could associate with AP2 protein. Endogenous c-KIT gene expression levels were elevated in AP2 stably-transfected human melanoma A375SM cells. Expression of exogenous AP2 in A375SM cells inhibited their tumorigenicity and metastatic potential in nude mice. The c-KIT ligand, SCF, also induced apoptosis in the AP2 stably-transfected A375SM cells. The identification of AP2 as an important regulator for c-KIT expression suggests that AP2 may have tumor growth and metastasis inhibitory properties, possibly mediated through c-KIT/SCF effects on apoptosis of human melanoma cells. Since AP2 binding sites were found in the promoters of other genes involved in the progression of human melanoma, such as MMP2 (72 kDa collagenase), MCAM/MUC18 and P21/WAF-1, our findings suggest that loss of AP2 expression might be a crucial event in the development of malignant melanoma. ^

Radioactivity of the Chernobyl fallout at Tyrolean glaciers

The stratigraphy of the gross-beta-activity of the Chernobyl fallout was measured in samples from glaciers in Tyrol. Great regional differences were obtained. The comparison of the results of samples collected in 1986 and 1987 shows a significant immigration of the fission products into deeper layers by melt water percolation but without any significant fractionation occuring. Gammaspectra show the dominance of 137Cs with the ratio 137Cs/134Cs of 2.6 to 2.9 (measurement February 1987) and 3.7 (measurement January 1988).

Alkenone analyses of sediment cores from the western Arabian Sea

Records of total organic carbon (TOC) and C37 alkenones were used as indicators for past primary productivity in the western and eastern Arabian Sea. Data from GeoB 3005, an open ocean site in the western Arabian Sea upwelling area, are compared with similar records of GeoB 3007 from the Owen Ridge, Ocean Drilling Program (ODP) Site 723 from the continental margin off Oman and MD 900963 from the eastern Arabian Sea. TOC/C37 alkenone records together with other proxies used to reconstruct upwelling intensity, indicate periods of high productivity in tune with precessional forcing all over the Arabian Sea. Based on their phase-relationship to variations in boreal summer insolation they can be divided into three groups. In the western Arabian Sea the precession-related phasing is different between productivity proxies and those for summer monsoon wind strength and upwelling intensity. TOC and C37 alkenone records from the western Arabian Sea lag the other monsoonal indicators by about 5 kyr, but lead productivity indicators from the eastern Arabian Sea by 3 kyr. Based on the differences in phase relationships associated with the precessional cycling between productivity and monsoonal proxies in the western Arabian Sea it is proposed that the TOC/C37 alkenone signal in the western Arabian Sea document a combined signal of moderate SW monsoon winds and of strengthened and prolonged NE monsoon winds. In the eastern Arabian Sea the phasing hints to coincidence between maximum productivity and stronger NE monsoon winds associated with precession-related maxima in ice volume. In contrast, variations in paleoproductivity at site GeoB 3007 from the Owen Ridge indicate productivity maxima during glacial substages 8.2, 6.2 and 2.2, whereas precessionrelated changes are of only minor importance at this location. The results of frequency analyses confirm that productivity at site GeoB 3007 responds predominantly to glacialinterglacial climate changes, while site GeoB 3005 from the open ocean upwelling region near the Gulf of Aden is dominated by precessional insolation. A possible explanation for the pattern revealed at the Owen Ridge is the periodic NW-SE displacement of the Findlater Jet axis, which separates the region of open ocean upwelling to the northwest from downwelling to the southeast ofthe jet. The carbon isotopes of planktic foraminifera reflect nutrient related d13C variations of dissolved inorganic carbon. The difference between the planktic foraminifera Globigerinoides ruber (w), living in the upper 50 m of the water column, and the deeper Iiving Neogloboquadrina dutertrei (Delta d13Cr-d) of core GeoB 3005 displays nutrient variations in the upwelling area near the Gulf of Aden. The results of cross-spectral analyses between Deltad13Cr-d of GeoB 3005 and proxies for SW monsoon intensity indicate, too, a dissociation of productivity from monsoonal upwelling intensity. Instead, productivity depends mainly on the availability of nutrients, while upwelling intensity of sub-surface water masses seems to be of only secondary importance. Additionally, sea surface temperatures (SSTs) were reconstructed using the unsaturation ratio of C37 alkenones. Alkenone SSTs reflect annual mean temperatures rather than explicitly the season of upwelling. This is evident from alkenone SSTs in a transect of surface sediments extending from the inner Gulf of Aden into the western Arabian Sea. The alkenone-derived SST records of GeoB 3005 from the open ocean upwelling region near the Gulf of Aden and GeoB 3007 from the Owen Ridge reveal similar variations with high SSTs during interglacial and low SSTs during glacial periods. The glacial marine oxygen isotope stage (MIS) 6 remains relatively warm and was not as cold as MIS 3 to 4 and 8 according to the alkenone SST. Similar variation-patterns were reconstructed in the coastal upwelling area off Oman for ODP Site 723 as weIl as in the eastern Arabian Sea for MD 900963, where upwelling is not as pronounced as in the western Arabian Sea. Spectral-analyses indicate that SST changes are in good agreement with the modulation of low-latitude precessional insolation changes by eccentricity. However, they do not show the pronounced cydicity in the precessional frequency band, which is characteristic for variations in paleoproductivity. Although the overall variation pattern is very similar, a dose comparison between the western (GeoB 3005) and the eastern Arabian Sea (MD 900963) shows larger differences between both sites during cold intervals than during periods of warm SSTs. This is attributed to a more effective cooling of surface waters in the western Arabian Sea by prolonged NE monsoon winds during times of expanded Northern Hemisphere ice-sheets, thereby lowering the annual mean SSTs stronger than in the eastern Arabian Sea.

Historia General de España

Sign. : []2, [asterisco]-2[asterisco]2, A-Z4, 2A-2D4, 2E2

Hibridación termosolar en centrales convencionales de generación de electricidad (HiTERCON)

El modelo energético actual está marcado por el predominio de los combustibles fósiles y el intento de introducir energías renovables para evolucionar hacia un modelo más sostenible. Entre las distintas alternativas, las plantas híbridas solar-fósil parecen una solución interesante ya que combinan las ventajas de las dos fuentes de energía. Hasta el momento, las líneas de investigación y análisis de plantas híbridas se ha centrado en el acoplamiento de tecnología solar a plantas o ciclos de potencia operativos. Este enfoque, tiene sentido en un marco como el actual, donde las plantas híbridas operativas no disponen de sistema de almacenamiento y donde se intenta reducir los costes, acoplando tecnología solar a ciclos de potencia existentes. Sin embargo, resta generalidad al análisis de la hibridación, imponiendo el diseño de la planta. Resulta interesante por tanto, realizar estudios que analicen la hibridación desde un punto de vista más general y que permitan a la propia planta, optimizar su diseño. Este estudio pretende aportar conocimiento en esta línea, analizando distintas posibilidades de diseño de futuras plantas híbridas operando en modo Fuel saving mode (FSM). Para ello, se ha realizado un estudio general y sistemático de distintas topologías de plantas híbridas, las cuales consideran como ciclo de potencia el ciclo Brayton y el ciclo Rankine. Para aportar la máxima generalidad posible, se han considerado un amplio abanico de condiciones operativas en ambos bloques. Como aporte al resto de estudios, éste estudio analiza la hibridación desde el punto de vista del bloque termosolar lo que indirectamente permite analizar el funcionamiento de la planta en modo híbrido. Para ello, se han identificado dos casos de diseño: 1. Caso A: Diseño de planta híbrida a partir de una planta o bloque convencional 2. Caso B: Diseño de planta híbrida a partir de una tecnología solar determinada La distinción entre casos de diseño, permite por un lado, establecer el potencial de la hibridación en las dos situaciones que se pueden plantear en la práctica estableciendo además, una base comparativa de resultados y por otro lado, identificar las condiciones de funcionamiento óptimas en las que diseñar la planta. Como conclusión principal, el estudio realizado demuestra que las condiciones de funcionamiento que optimizan la planta híbrida pueden diferir dependiendo del caso de diseño que se considere, lo que implica que el funcionamiento de la planta no tiene porqué estar optimizado simultáneamente para sus dos modos de funcionamiento (modo fósil y modo híbrido). Por tanto, se considera que el diseño final de la planta híbrida debería estar en función del tiempo en que la planta opera en cada uno de los modos, es decir, del Solar Capacity Factor (SCF). Además, el estudio realizado identifica dos situaciones concretas para las cuales la hibridación estaría desaconsejada desde un punto de vista energético. Aunque los resultados obtenidos son sólo una primera aproximación del diseño final de la planta híbrida, proporcionan unas directrices a partir de la cuales iniciar su desarrollo. Esto es especialmente interesante de cara a políticas estratégicas, donde en función de los objetivos vigentes o que se puedan plantear, se puede modificar el diseño de la planta en su estructura básica.

Ubiquitin-dependent degradation of IκBα is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1

Activation of the transcription factor nuclear factor kappa B (NF-κB) is controlled by proteolysis of its inhibitory subunit (IκB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IκBα by a large multisubunit complex containing IκB kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/βTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IκBα only when IκBα is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IκBα in concert with IκB kinases, resulting in nuclear translocation of NF-κB. In addition, FWD1 strikingly evoked the ubiquitination of IκBα in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IκBα. These results suggest that the substrate-specific degradation of IκBα is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated IκBα. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-κB through control of IκB protein stability.

Chymase cleavage of stem cell factor yields a bioactive, soluble product

Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.

Phosphorylation Controls Timing of Cdc6p Destruction: A Biochemical Analysis

The replication initiation protein Cdc6p forms a tight complex with Cdc28p, specifically with forms of the kinase that are competent to promote replication initiation. We now show that potential sites of Cdc28 phosphorylation in Cdc6p are required for the regulated destruction of Cdc6p that has been shown to occur during the Saccharomyces cerevisiae cell cycle. Analysis of Cdc6p phosphorylation site mutants and of the requirement for Cdc28p in an in vitro ubiquitination system suggests that targeting of Cdc6p for degradation is more complex than previously proposed. First, phosphorylation of N-terminal sites targets Cdc6p for polyubiquitination probably, as expected, through promoting interaction with Cdc4p, an F box protein involved in substrate recognition by the Skp1-Cdc53-F-box protein (SCF) ubiquitin ligase. However, in addition, mutation of a single, C-terminal site stabilizes Cdc6p in G2 phase cells without affecting substrate recognition by SCF in vitro, demonstrating a second and novel requirement for specific phosphorylation in degradation of Cdc6p. SCF-Cdc4p– and N-terminal phosphorylation site–dependent ubiquitination appears to be mediated preferentially by Clbp/Cdc28p complexes rather than by Clnp/Cdc28ps, suggesting a way in which phosphorylation of Cdc6p might control the timing of its degradation at then end of G1 phase of the cell cycle. The stable cdc6 mutants show no apparent replication defects in wild-type strains. However, stabilization through mutation of three N-terminal phosphorylation sites or of the single C-terminal phosphorylation site leads to dominant lethality when combined with certain mutations in the anaphase-promoting complex.

Chimeric Cytokine Receptor Can Transduce Expansion Signals in Interleukin 6 Receptor α (IL-6Rα)-, IL-11Rα-, and gp130-low to -negative Primitive Hematopoietic Progenitors

We generated transgenic mice expressing chimeric receptors, which comprise extracellular domains of the human granulocyte–macrophage colony-stimulating factor (hGM-CSF) receptor and transmembrane and cytoplasmic domains of the mouse leukemia inhibitory factor receptor. In suspension cultures of lineage-negative (Lin−), 5-fluorouracil-resistant bone marrow cells of the transgenic mice, a combination of hGM-CSF and stem cell factor (SCF) induced exponential expansions of mixed colony-forming unit. The combination of hGM-CSF and SCF was effective on enriched, Lin−Sca-1+c-kit+ progenitors and increased either mixed colony-forming unit or cobblestone area–forming cells. In case of stimulation with hGM-CSF and SCF, interleukin-6 (IL-6) and SCF, or IL-11 and SCF, the most efficient expansion was achieved with hGM-CSF and SCF. When Lin−Sca-1+c-kit+CD34− further enriched progenitors were clone sorted and individually incubated in the presence of SCF, hGM-CSF stimulated a larger number of cells than did IL-6, IL-6 and soluble IL-6 receptor (IL-6R), or IL-11. These data suggest the presence of IL-6Rα-, IL-11Rα-, and gp130-low to -negative primitive hematopoietic progenitors. Such primitive progenitors are equipped with signal transduction molecules and can expand when these chimeric receptors are genetically introduced into the cells and stimulated with hGM-CSF in the presence of SCF.

Activation of HIF1α ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex

Mutations in the VHL tumor suppressor gene result in constitutive expression of many hypoxia-inducible genes, at least in part because of increases in the cellular level of hypoxia-inducible transcription factor HIF1α, which in normal cells is rapidly ubiquitinated and degraded by the proteasome under normoxic conditions. The recent observation that the VHL protein is a subunit of an Skp1-Cul1/Cdc53-F-box (SCF)-like E3 ubiquitin ligase raised the possibility that VHL may be directly responsible for regulating cellular levels of HIF1α by targeting it for ubiquitination and proteolysis. In this report, we test this hypothesis directly. We report development of methods for production of the purified recombinant VHL complex and present direct biochemical evidence that it can function with an E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzyme to activate HIF1α ubiquitination in vitro. Our findings provide new insight into the function of the VHL tumor suppressor protein, and they provide a foundation for future investigations of the mechanisms underlying VHL regulation of oxygen-dependent gene expression.

Functions of the DNA damage response pathway target Ho endonuclease of yeast for degradation via the ubiquitin-26S proteasome system

Ho endonuclease of Saccharomyces cerevisiae is a homing endonuclease that makes a site-specific double-strand break in the MAT gene in late G1. Here we show that Ho is rapidly degraded via the ubiquitin-26S proteasome system through two ubiquitin-conjugating enzymes UBC2Rad6 and UBC3Cdc34. UBC2Rad6 is complexed with the ring finger DNA-binding protein Rad18, and we find that Ho is stabilized in rad18 mutants. We show that the Ho degradation pathway involving UBC3Cdc34 goes through the Skp1/Cdc53/F-box (SCF) ubiquitin ligase complex and identify a F-box protein, Yml088w, that is required for Ho degradation. Components of a defined pathway of the DNA damage response, MEC1, RAD9, and CHK1, are also necessary for Ho degradation, whereas functions of the RAD24 epistasis group and the downstream effector RAD53 have no role in degradation of Ho. Our results indicate a link between the endonuclease function of Ho and its destruction.

Lineage commitment in the progeny of murine hematopoietic preprogenitor cells: Influence of thrombopoietin and interleukin 5

Normal mouse marrow cells were stimulated by stem cell factor (SCF) to form dispersed or multicentric blast colonies containing progenitor cells committed to various hematopoietic lineages. Combination of the eosinophil-specific regulator interleukin 5 with SCF increased the frequency of colonies containing eosinophil-committed progenitor cells with multicentric but not dispersed blast colonies. Combination of thrombopoietin with SCF increased the frequency of colonies containing megakaryocyte-committed progenitor cells with both types of blast colony. Neither interleukin 5 nor thrombopoietin significantly altered the number or total cell content of blast colonies or progenitor cell numbers in blast colonies from those stimulated by SCF alone. No correlation was observed between total progenitor cell content and the presence or absence of either eosinophil or megakaryocyte progenitors in either type of blast colony. The data argue against a random process as being responsible for the formation of particular committed progenitor cells or the possibility that lineage-specific regulators merely enhance survival of such committed progenitor cells formed in developing blast colonies.

Skp2 is oncogenic and overexpressed in human cancers

Skp2 is a member of the F-box family of substrate-recognition subunits of SCF ubiquitin–protein ligase complexes that has been implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G1 progression, including the cyclin-dependent kinase inhibitor p27, a dosage-dependent tumor suppressor protein. In this study, we examined Skp2 and p27 protein expression by immunohistochemistry in normal oral epithelium and in different stages of malignant oral cancer progression, including dysplasia and oral squamous cell carcinoma. We found that increased levels of Skp2 protein are associated with reduced p27 in a subset of oral epithelial dysplasias and carcinomas compared with normal epithelial controls. Tumors with high Skp2 (>20% positive cells) expression invariably showed reduced or absent p27 and tumors with high p27 (>20% positive cells) expression rarely showed Skp2 positivity. Increased Skp2 protein levels were not always correlated with increased cell proliferation (assayed by Ki-67 staining), suggesting that alterations of Skp2 may contribute to the malignant phenotype without affecting proliferation. Skp2 protein overexpression may lead to accelerated p27 proteolysis and contribute to malignant progression from dysplasia to oral epithelial carcinoma. Moreover, we also demonstrate that Skp2 has oncogenic potential by showing that Skp2 cooperates with H-RasG12V to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice. The observations that Skp2 can mediate transformation and is up-regulated during oral epithelial carcinogenesis support a role for Skp2 as a protooncogene in human tumors.

Protacs: Chimeric molecules that target proteins to the Skp1–Cullin–F box complex for ubiquitination and degradation

The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One of the best-characterized enzymes that catalyzes the attachment of ubiquitin to proteins is a ubiquitin ligase complex, Skp1-Cullin-F box complex containing Hrt1 (SCF). We sought to artificially target a protein to the SCF complex for ubiquitination and degradation. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds the angiogenesis inhibitor ovalicin. A chimeric compound, protein-targeting chimeric molecule 1 (Protac-1), was synthesized to recruit MetAP-2 to SCF. One domain of Protac-1 contains the IκBα phosphopeptide that is recognized by the F-box protein β-TRCP, whereas the other domain is composed of ovalicin. We show that MetAP-2 can be tethered to SCFβ-TRCP, ubiquitinated, and degraded in a Protac-1-dependent manner. In the future, this approach may be useful for conditional inactivation of proteins, and for targeting disease-causing proteins for destruction.