Non-small cell lung cancer (NSCLC) is a prevalent and devastating disease that claims more lives than breast, prostate, colon and pancreatic cancers combined. Current research suggests that standard ...chemotherapy regimens have been optimized to maximal efficiency. Promising new treatment strategies involve novel agents targeting molecular aberrations present in subsets of NSCLC. We evaluated 88 human NSCLC tumors of diverse histology and identified Mer and Axl as receptor tyrosine kinases (RTKs) overexpressed in 69% and 93%, respectively, of tumors relative to surrounding normal lung tissue. Mer and Axl were also frequently overexpressed and activated in NSCLC cell lines. Ligand-dependent Mer or Axl activation stimulated MAPK, AKT and FAK signaling pathways indicating roles for these RTKs in multiple oncogenic processes. In addition, we identified a novel pro-survival pathway-involving AKT, CREB, Bcl-xL, survivin, and Bcl-2-downstream of Mer, which is differentially modulated by Axl signaling. We demonstrated that short hairpin RNA (shRNA) knockdown of Mer or Axl significantly reduced NSCLC colony formation and growth of subcutaneous xenografts in nude mice. Mer or Axl knockdown also improved in vitro NSCLC sensitivity to chemotherapeutic agents by promoting apoptosis. When comparing the effects of Mer and Axl knockdown, Mer inhibition exhibited more complete blockade of tumor growth while Axl knockdown more robustly improved chemosensitivity. These results indicate that Mer and Axl have complementary and overlapping roles in NSCLC and suggest that treatment strategies targeting both RTKs may be more effective than singly-targeted agents. Our findings validate Mer and Axl as potential therapeutic targets in NSCLC and provide justification for development of novel therapeutic compounds that selectively inhibit Mer and/or Axl.
Abstract Recent evidence demonstrates that N -methyl- d -aspartate receptor (NMDAR) trafficking contributes to synaptic plasticity in the hippocampus. Phosphorylation of tyrosine residues, especially ...NR2B tyrosine 1472, appears to be a mechanism by which NMDAR endocytosis is prevented, suggesting that the tyrosine phosphorylation and surface expression of NMDARs are positively correlated. Previous work from our laboratory and others has confirmed that modulation of tyrosine phosphatase and kinase activity alters the surface expression of NMDARs. However, the changes in NMDAR surface expression described in those studies were in terms of total surface membrane versus intracellular receptors. Within the plasma membrane of glutamatergic synapses, distinct populations of NMDARs exist. Namely, receptors at the surface can be differentiated into synaptic and extrasynaptic pools based on their association with the post-synaptic density (PSD) and availability to glutamate. In the present study, we utilized a subcellular fractionation approach coupled with detergent extraction to prepare synaptic and extrasynaptic NMDARs from adult rat hippocampal slices. Using this method, we examined how tyrosine phosphatase and Src-family tyrosine kinase (SFK) inhibitors modulate the phosphorylation and localization of these different pools of NMDARs. We found that both synaptic and extrasynaptic NMDARs were modulated by tyrosine phosphatase and SFK inhibitors; however subunit- and residue-specific effects were observed. Specifically, phosphorylation of NR2B tyrosine 1472 was associated with enrichment of synaptic NMDARs, whereas phosphorylation of NR2B tyrosine 1336 was associated with enrichment of extrasynaptic NMDARs. Using electrophysiological methods, we also reveal that the biochemical modifications produced by these inhibitors were associated with corresponding changes in NMDAR function.
The rapid evolution of telomere proteins has hindered identification of orthologs from diverse species and created the impression that certain groups of eukaryotes have largely non-overlapping sets ...of telomere proteins. However, the recent identification of additional telomere proteins from various model organisms has dispelled this notion by expanding our understanding of the composition, architecture and range of telomere protein complexes present in individual species. It is now apparent that versions of the budding yeast CST complex and mammalian shelterin are present in multiple phyla. While the precise subunit composition and architecture of these complexes vary between species, the general function is often conserved. Despite the overall conservation of telomere protein complexes, there is still considerable species-specific variation, with some organisms having lost a particular subunit or even an entire complex. In some cases, complex components appear to have migrated between the telomere and the telomerase RNP. Finally, gene duplication has created telomere protein paralogs with novel functions. While one paralog may be part of a conserved telomere protein complex and have the expected function, the other paralog may serve in a completely different aspect of telomere biology.
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Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The New World screwworm fly, Cochliomyia hominivorax, and the Australian sheep blow fly, Lucilia cuprina, are major pests of livestock. The sterile insect technique was used to eradicate C. ...hominivorax from North and Central America. This involved area‐wide releases of male and female flies that had been sterilized by radiation. Genetic systems have been developed for making ‘male‐only’ strains that would improve the efficiency of genetic control of insect pests. One system involves induction of female lethality in embryos through activation of a pro‐apoptotic gene by the tetracycline‐dependent transactivator. Sex‐specific expression is achieved using an intron from the transformer gene, which we previously isolated from several calliphorids. In the present study, we report the isolation of the promoters from the C. hominivorax slam and Lucilia sericata bnk cellularization genes and show that these promoters can drive expression of a GFP reporter gene in early embryos of transgenic L. cuprina. Additionally, we report the isolation of the L. sericata pro‐apoptotic hid and rpr genes, identify conserved motifs in the encoded proteins and determine the relative expression of these genes at different stages of development. We show that widespread expression of the L. sericata pro‐apoptotic genes was lethal in Drosophila melanogaster. The isolated gene promoters and pro‐apoptotic genes could potentially be used to build transgenic embryonic sexing strains of calliphorid livestock pests.
Resistance to tobacco mosaic virus (TMV) is controlled by the single dominant gene N in Nicotiana glutinosa L. This gene has been transferred to cultivated tobacco (N. tabacum L.) by interspecific ...hybridization and backcrossing, but has historically been associated with reduced yields and/or quality in flue-cured tobacco breeding materials. Past researchers have suggested the role of pleiotropy and/or linkage drag effects in this unfavorable relationship. Introduction of the cloned N gene into a TMV-susceptible tobacco genotype (cultivar 'K326') via plant transformation permitted investigation of the relative importance of these possibilities. On average, yield and cash return ($ ha-¹) of 14 transgenic NN lines of K326 were significantly higher relative to an isoline of K326 carrying N introduced via interspecific hybridization and backcrossing. The negative effects of tissue culture-induced genetic variation confounded comparisons with the TMV-susceptible cultivar, K326, however. Backcrossing the original transgenic lines to non-tissue cultured K326 removed many of these unfavorable effects, and significantly improved their performance for yield and cash return. Comparisons of the 14 corresponding transgenic NN backcross-derived lines with K326 indicated that linkage drag is the main factor contributing to reduced yields in TMV-resistant flue-cured tobacco germplasm. On average, these transgenic lines outyielded the conventionally-developed TMV-resistant K326 isoline by 427 kg ha-¹ (P < 0.05) and generated $1,365 ha-¹ more (P < 0.05). Although transgenic tobacco cultivars are currently not commercially acceptable, breeding strategies designed to reduce the amount of N. glutinosa chromatin linked to N may increase the likelihood of developing high-yielding TMV-resistant flue-cured tobacco cultivars.
Tetrahymena telomeres are protected by a protein complex composed of Pot1, Tpt1, Pat1, and Pat2. Pot1 binds the 3' overhang and serves multiple roles in telomere maintenance. Here we describe Pot2, a ...paralog of Pot1 which has evolved a novel function during Tetrahymena sexual reproduction. Pot2 is unnecessary for telomere maintenance during vegetative growth, as the telomere structure is unaffected by POT2 macronuclear gene disruption. Pot2 is expressed only in mated cells, where it accumulates in developing macronuclei around the time of two chromosome processing events: internal eliminated sequence (IES) excision and chromosome breakage. Chromatin immunoprecipitation (ChIP) demonstrated Pot2 localization to regions of chromosome breakage but not to telomeres or IESs. Pot2 association with chromosome breakage sites (CBSs) occurs slightly before chromosome breakage. Pot2 did not bind CBSs or telomeric DNA in vitro, suggesting that it is recruited to CBSs by another factor. The telomere proteins Pot1, Pat1, and Tpt1 and the IES binding factor Pdd1 fail to colocalize with Pot2. Thus, Pot2 is the first protein found to associate specifically with CBSs. The selective association of Pot2 versus Pdd1 with CBSs or IESs indicates a mechanistic difference between the chromosome processing events at these two sites. Moreover, ChIP revealed that histone marks characteristic of IES processing, H3K9me3 and H3K27me3, are absent from CBSs. Thus, the mechanisms of chromosome breakage and IES excision must be fundamentally different. Our results lead to a model where Pot2 directs chromosome breakage by recruiting telomerase and/or the endonuclease responsible for DNA cleavage to CBSs.
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