Inactivation of V79 cells by low-energy protons, deuterons and helium-3 ions

Previous work by ourselves and by others has demonstrated that protons with a linear energy transfer (LET) about 30 V mu m(-1) are more effective at killing cells than doubly charged particles of the same LET. In this work we show that by using deuterons, which have about twice the range of protons with the same LET, it is possible to extend measurements of the RBE of singly charged particles to higher LET (up to 50 keV mu m(-1)). We report the design and use of a new arrangement for irradiating V79 mammalian cells. Cell survival. measurements have been made using protons in the energy range 1.0-3.7 MeV, deuterons in the energy range 0.9-3.4 MeV and He-3(2+) ions in the energy range 3.4-6.9 MeV;. This corresponds to volume-averaged LET (within the cell nucleus) between 10 and 28 keV mu m(-1) for protons, 18-50 keV mu m(-1) for deuterons, and 59-106 keV mu m(-1) for helium ions. Our results show no difference in the effectiveness of protons and deuterons matched for LET. However, for LET above about 30 keV mu m(-1) singly charged particles are more effective at inactivating cells than doubly-charged particles of the same LET and that this difference can be understood in terms of the radial dose distribution around the primary ion track.

Genomic instability in Chinese hamster cells after exposure to X rays or alpha particles of different mean linear energy transfer

Evidence has accumulated that radiation induces a transmissible persistent destabilization of the genome, which mag. result in effects arising in the progeny of irradiated but surviving cells. An enhanced death rate among the progeny of cells surviving irradiation persists for many generations in the form of a reduced plating efficiency. Such delayed reproductive death is correlated with an increased occurrence of micronuclei. Since it has been suggested that radiation-induced chromosomal instability might depend on the radiation quality, we investigated the effects of alpha particles of different LET by looking at the frequency of delayed micronuclei in Chinese hamster V79 cells after cytochalasin-induced block of cell division, A dose-dependent increase in the frequency of micronuclei was found in cells assayed 1 week postirradiation or later. Also, there was a persistent increase in the frequency of dicentrics in surviving irradiated cells, Moreover, we found an increased micronucleus frequency in all of the 30 clones isolated from individual cells which had been irradiated with doses equivalent to either one, two or three alpha-particle traversals per cell nucleus, We conclude that the target for genomic instability in Chinese hamster cells must be larger than the cell nucleus. (C) 1997 by Radiation Research Society

Distinct CCK-2 Receptor Conformations Associated with β-Arrestin-2 Recruitment or Phospholipase-C Activation Revealed by a Biased Antagonist

Seven-transmembrane receptors (7TMRs), also termed G protein-coupled receptors (GPCRs), form the largest class of cell surface membrane receptors, involving several hundred members in the human genome. Near 30% of marketed pharmacological agents target 7TMRs. 7TMRs adopt multiple conformations upon agonist binding. Biased agonists, in contrast to non-biased agonists, are believed to stabilize conformations preferentially activating either G-protein- or ß-arrestin-dependent signalling pathways. However, proof that cognate conformations of receptors display structural differences within their binding site where biased agonism initiates, are still lacking. Here, we show that a non-biased agonist, cholecystokinin (CCK) induces conformational states of the CCK2R activating Gq-protein-dependent pathway (CCK2RG) or recruiting ß-arrestin2 (CCK2Rß) that are pharmacologically and structurally distinct. Two structurally unrelated antagonists competitively inhibited both pathways. A third ligand (GV150,013X), acted as a high affinity competitive antagonist on CCK2RG but was nearly inefficient as inhibitor of CCK2Rß. Several structural elements on both GV150,013X and in CCK2R binding cavity, which hinder binding of GV150,013X only to the CCK2Rß were identified. At last, proximity between two conserved amino acids from transmembrane helices 3 and 7 interacting through sulphur-aromatic interaction was shown to be crucial for selective stabilization of the CCK2Rß state. These data establish structural evidences for distinct conformations of a 7TMR associated with ß-arrestin-2 recruitment or G-protein coupling and validate relevance of the design of biased ligands able to selectively target each functional conformation of 7TMRs.

Direct reprogramming of fibroblasts into endothelial cells capable of angiogenesis and reendothelialization in tissue-engineered vessels

The generation of induced pluripotent stem (iPS) cells is an important tool for regenerative medicine. However, the main restriction is the risk of tumor development. In this study we found that during the early stages of somatic cell reprogramming toward a pluripotent state, specific gene expression patterns are altered. Therefore, we developed a method to generate partial-iPS (PiPS) cells by transferring four reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) to human fibroblasts for 4 d. PiPS cells did not form tumors in vivo and clearly displayed the potential to differentiate into endothelial cells (ECs) in response to defined media and culture conditions. To clarify the mechanism of PiPS cell differentiation into ECs, SET translocation (myeloid leukemia-associated) (SET) similar protein (SETSIP) was indentified to be induced during somatic cell reprogramming. Importantly, when PiPS cells were treated with VEGF, SETSIP was translocated to the cell nucleus, directly bound to the VE-cadherin promoter, increasing vascular endothelial-cadherin (VE-cadherin) expression levels and EC differentiation. Functionally, PiPS-ECs improved neovascularization and blood flow recovery in a hindlimb ischemic model. Furthermore, PiPS-ECs displayed good attachment, stabilization, patency, and typical vascular structure when seeded on decellularized vessel scaffolds. These findings indicate that reprogramming of fibroblasts into ECs via SETSIP and VEGF has a potential clinical application.

Pigment epithelium-derived factor mitigates inflammation and oxidative stress in retinal pericytes exposed to oxidized low-density lipoprotein

Oxidized and/or glycated low-density lipoprotein (LDL) may mediate capillary injury in diabetic retinopathy. The mechanisms may involve pro-inflammatory and pro-oxidant effects on retinal capillary pericytes. In this study, these effects, and the protective effects of pigment epithelium-derived factor (PEDF), were defined in a primary human pericyte model. Human retinal pericytes were exposed to 100 microg/ml native LDL (N-LDL) or heavily oxidized glycated LDL (HOG-LDL) with or without PEDF at 10-160 nM for 24 h. To assess pro-inflammatory effects, monocyte chemoattractant protein-1 (MCP-1) secretion was measured by ELISA, and nuclear factor-kappaB (NF-kappaB) activation was detected by immunocytochemistry. Oxidative stress was determined by measuring intracellular reactive oxygen species (ROS), peroxynitrite (ONOO(-)) formation, inducible nitric oxide synthase (iNOS) expression, and nitric oxide (NO) production. The results showed that MCP-1 was significantly increased by HOG-LDL, and the effect was attenuated by PEDF in a dose-dependent manner. PEDF also attenuated the HOG-LDL-induced NF-kappaB activation, suggesting that the inhibitory effect of PEDF on MCP-1 was at least partially through the blockade of NF-kappaB activation. Further studies demonstrated that HOG-LDL, but not N-LDL, significantly increased ONOO(-) formation, NO production, and iNOS expression. These changes were also alleviated by PEDF. Moreover, PEDF significantly ameliorated HOG-LDL-induced ROS generation through up-regulation of superoxide dismutase 1 expression. Taken together, these results demonstrate pro-inflammatory and pro-oxidant effects of HOG-LDL on retinal pericytes, which were effectively ameliorated by PEDF. Suppressing MCP-1 production and thus inhibiting macrophage recruitment may represent a new mechanism for the salutary effect of PEDF in diabetic retinopathy and warrants more studies in future.

DNA methyltransferase expression in the mouse germ line during periods of de novo methylation

DNA methyltransferase (DNMT) 3A and DNMT3B are both active de novo DNA methyltransferases required for development, whereas DNMT3L, which has no demonstrable methyltransferase activity, is required for methylation of imprinted genes in the oocyte. We show here that different mechanisms are used to restrict access by these proteins to their targets during germ cell development. Transcriptional control of the Dnmt3l promoter guarantees that message is low or absent except during periods of de novo activity. Use of an alternative promoter at the Dnmt3a locus produces the shorter Dnmt3a2 transcript in the germ line and postimplantation embryo only, whereas alternative splicing of the Dnmt3b transcript ensures that Dnmt3b1 is absent in the male prospermatogonia. Control of subcellular protein localization is a common theme for DNMT3A and DNMT3B, as proteins were seen in the nucleus only when methylation was occurring. These mechanisms converge to ensure that the only time that functional products from each locus are present in the germ cell nuclei is around embryonic day 17.5 in males and after birth in the growing oocytes in females.

Intracellular antigenic changes associated with meiosis in the orthopteran Stauroderus scalaris

Monoclonal antibodies have been prepared against purified pachytene cells from grasshopper testes. Immunoblotting and immunofluorescence analyses identified those monoclonal antibodies which showed specificity for antigens in pachytene cells. Several antigenic changes were found to be associated with meiotic cells. Five monoclonal antibodies detected antigens which were located in the cytoplasm of premeiotic cells but were nuclear during meiosis. One monoclonal antibody showed a discrete cytoplasmic fluorescent pattern in meiotic, but not in premeiotic, cells. Another bound specifically to the nuclei of some epithelial cells at the base of follicles in mature testes.

Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of PI 3-kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella containing vacuolae (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV-killed bacteria, the majority of live bacteria did not colocalize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K-Akt-Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the downregulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation which may contribute to Klebsiella pathogenesis.

Alpha particles induce pan-nuclear phosphorylation of H2AX in primary human lymphocytes mediated through ATM

The use of high linear energy transfer radiations in the form of carbon ions in heavy ion beam lines or alpha particles in new radionuclide treatments has increased substantially over the past decade and will continue to do so due to the favourable dose distributions they can offer versus conventional therapies. Previously it has been shown that exposure to heavy ions induces pan-nuclear phosphorylation of several DNA repair proteins such as H2AX and ATM in vitro. Here we describe similar effects of alpha particles on ex vivo irradiated primary human peripheral blood lymphocytes. Following alpha particle irradiation pan-nuclear phosphorylation of H2AX and ATM, but not DNA-PK and 53BP1, was observed throughout the nucleus. Inhibition of ATM, but not DNA-PK, resulted in the loss of pan-nuclear phosphorylation of H2AX in alpha particle irradiated lymphocytes. Pan-nuclear gamma-H2AX signal was rapidly lost over 24h at a much greater rate than foci loss. Surprisingly, pan-nuclear gamma-H2AX intensity was not dependent on the number of alpha particle induced double strand breaks, rather the number of alpha particles which had traversed the cell nucleus. This distinct fluence dependent damage signature of particle radiation is important in both the fields of radioprotection and clinical oncology in determining radionuclide biological dosimetry and may be indicative of patient response to new radionuclide cancer therapies.

Nuclear translocation and functions of growth factor receptors

Cellular signal transduction in response to environmental signals involves a relay of precisely regulated signal amplifying and damping events. A prototypical signaling relay involves ligands binding to cell surface receptors and triggering the activation of downstream enzymes to ultimately affect the subcellular distribution and activity of DNA-binding proteins that regulate gene expression. These so-called signal transduction cascades have dominated our view of signaling for decades. More recently evidence has accumulated that components of these cascades can be multifunctional, in effect playing a conventional role for example as a cell surface receptor for a ligand whilst also having alternative functions for example as transcriptional regulators in the nucleus. This raises new challenges for researchers. What are the cues/triggers that determine which role such proteins play? What are the trafficking pathways which regulate the spatial distribution of such proteins so that they can perform nuclear functions and under what circumstances are these alternative functions most relevant?

Nuclear LYRIC/AEG-1 interacts with PLZF and relieves PLZF-mediated repression

LYRIC/AEG-1 and its altered expression have been linked to carcinogenesis in prostate, brain and melanoma as well as promoting chemoresistance and metastasis in breast cancer. LYRIC/AEG-1 function remains unclear, although LYRIC/AEG-1 is activated by oncogenic HA-RAS, through binding of c-myc to its promoter, which in turn regulates the key components of the PI3-kinase and nuclear factor-kappaB pathways. We have identified the transcriptional repressor PLZF as an interacting protein of LYRIC/AEG through a yeast two-hybrid screen. PLZF regulates the expression of genes involved in cell growth and apoptosis including c-myc. Coexpression of LYRIC/AEG-1 with PLZF leads to a reduction in PLZF-mediated repression by reducing PLZF binding to promoters. We have confirmed that nuclear LYRIC/AEG-1 and PLZF interact in mammalian cells via the N- and C termini of LYRIC/AEG-1 and a region C terminal to the RD2 domain of PLZF. Both proteins colocalize to nuclear bodies containing histone deacetylases, which are known to promote PLZF-mediated repression. Our data suggest one mechanism for cells with altered LYRIC/AEG-1 expression to evade apoptosis and increase cell growth during tumourigenesis through the regulation of PLZF repression.

LYRIC/AEG-1 is targeted to different subcellular compartments by ubiquitinylation and intrinsic nuclear localization signals

PURPOSE: LYRIC/AEG-1 has been reported to influence breast cancer survival and metastases, and its altered expression has been found in a number of cancers. The cellular function of LYRIC/AEG-1 has previously been related to its subcellular distribution in cell lines. LYRIC/AEG-1 contains three uncharacterized nuclear localization signals (NLS), which may regulate its distribution and, ultimately, function in cells.

EXPERIMENTAL DESIGN: Immunohistochemistry of a human prostate tissue microarray composed of 179 prostate cancer and 24 benign samples was used to assess LYRIC/AEG-1 distribution. Green fluorescent protein-NLS fusion proteins and deletion constructs were used to show the ability of LYRIC/AEG-1 NLS to target green fluorescent protein from the cytoplasm to the nucleus. Immunoprecipitation and Western blotting were used to show posttranslational modification of LYRIC/AEG-1 NLS regions.

RESULTS: Using a prostate tissue microarray, significant changes in the distribution of LYRIC/AEG-1 were observed in prostate cancer as an increased cytoplasmic distribution in tumors compared with benign tissue. These differences were most marked in high grade and aggressive prostate cancers and were associated with decreased survival. The COOH-terminal extended NLS-3 (amino acids 546-582) is the predominant regulator of nuclear localization, whereas extended NLS-1 (amino acids 78-130) regulates its nucleolar localization. Within the extended NLS-2 region (amino acids 415-486), LYRIC/AEG-1 can be modified by ubiquitin almost exclusively within the cytoplasm.

CONCLUSIONS: Changes in LYRIC/AEG-1 subcellular distribution can predict Gleason grade and survival. Two lysine-rich regions (NLS-1 and NLS-3) can target LYRIC/AEG-1 to subcellular compartments whereas NLS-2 is modified by ubiquitin in the cytoplasm.

Alterations in beta-catenin expression and localization in prostate cancer

BACKGROUND: Wnt signaling is thought to be important in prostate cancer, in part because proteins such as beta-catenin can also affect androgen receptor signaling. beta-Catenin forms a cell adhesion complex with E-cadherin raising the possibility that loss of expression or a change in beta-catenin distribution in the cell could also alter downstream signaling, decreased inter-cellular adhesion and the promotion of metastasis. A number of studies have reported the altered expression and/or localization of beta-catenin as a biomarker in prostate cancer.

METHODS: Tissue microarrays comprised of BPH and low, moderate and high-grade prostate cancer (n=77) were assessed for beta-catenin expression and distribution using immunohistochemistry. Staining was also performed on a tissue microarray containing tissue from patients before and after hormone manipulation. The effects of fixation and different antibodies was assessed on fixed LNCaP cell pellets and small prostate tissue microarrays.

RESULTS: We have observed increased beta-catenin expression in only high Gleason score (>7) prostate cancer. A nuclear re-distribution of beta-catenin has previously been reported. We noted nuclear beta-catenin in benign prostatic hyperplasia and a gradual loss in nuclear distribution with increasing Gleason grade. We found no evidence for an alteration in beta-catenin expression or re-distribution with hormone ablation. Altered fixation, antibodies and antibody concentration did affect the intensity and specificity of staining.

CONCLUSIONS: A loss of nuclear beta-catenin is the most consistent feature in prostate cancer rather than absolute levels of expression. We also suggest that variation in immunohistochemical protocols may explain variations in the reported literature.

Structural basis for the nuclear import of the human androgen receptor

Ligand-dependent nuclear import is crucial for the function of the androgen receptor (AR) in both health and disease. The unliganded AR is retained in the cytoplasm but, on binding 5alpha-dihydrotestosterone, it translocates into the nucleus and alters transcription of its target genes. Nuclear import of AR is mediated by the nuclear import factor importin-alpha, which functions as a receptor that recognises and binds to specific nuclear localisation signal (NLS) motifs on cargo proteins. We show here that the AR binds to importin-alpha directly, albeit more weakly than the NLS of SV40 or nucleoplasmin. We describe the 2.6-angstroms-resolution crystal structure of the importin-alpha-AR-NLS complex, and show that the AR binds to the major NLS-binding site on importin-alpha in a manner different from most other NLSs. Finally, we have shown that pathological mutations within the NLS of AR that are associated with prostate cancer and androgen-insensitivity syndrome reduce the binding affinity to importin-alpha and, subsequently, retard nuclear import; surprisingly, however, the transcriptional activity of these mutants varies widely. Thus, in addition to its function in the nuclear import of AR, the NLS in the hinge region of AR has a separate, quite distinct role on transactivation, which becomes apparent once nuclear import has been achieved.