Mechanisms of apoptosis in chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by the accumulation of terminally differentiated, mature B cells that do not progress beyond the G1 stage of cell cycle, suggesting that these cells possess intrinsic defects in apoptosis. Treatment relies heavily on chemotherapy (primarily nucleoside analogs and glucocorticoids) that may initially be effective in patients, but ultimately give rise to refractory, untreatable disease. The purpose of this study was to determine whether key components of the apoptotic machinery were intact in CLL lymphocytes, especially in patients refractory to therapy. ^ Activation of proteases has been shown to be at the core of the apoptotic pathway and this work demonstrates that protease activation is required for glucocorticoid and nucleoside analog-induced apoptosis in CLL cells. Inhibitors of serine proteases as well as caspase inhibitors blocked induced DNA fragmentation, and a peptide inhibitor of the nuclear scaffold (NS) protease completely suppressed both induced and spontaneous apoptosis. However, the NS protease inhibitor actually promoted several pro-apoptotic events, such as caspase activation, exposure of surface phosphatidylserine, and loss of mitochondrial membrane potential. These results suggested that the NS protease may interact with the apoptotic program in CLL cells at two separate points. ^ In order to further investigate the role of the NS protease in CLL, patient isolates were treated with proteasome inhibitors because of previous results suggesting that the ISIS protease might be a β subunit of the proteasome. Proteasome inhibitors induced massive DNA fragmentation in every patient tested, even in those resistant to the effects of glucocorticoid and nucleoside analogs in vitro. Several other features of apoptosis were also promoted by the proteasome inhibitor, including mitochondrial alterations such as release of cytochrome c and drops in mitochondrial membrane potential. Proteasome inhibitor-induced apoptosis was associated with inhibition of NFκB, a proteasome-regulated transcription factor that has been implicated in the suppression of apoptosis in a number of systems. The NS protease inhibitor also caused a decrease in active NFκB, suggesting that the proapoptotic effects of this agent might be due to depletion of NFκB. ^ Given these findings, the role of NFκB, in conferring survival in CLL was investigated. Glucocorticoid hormone treatment was shown to cause decreases in the activity of the transcription factor, while phorbol dibutyrate, which blocks glucocorticoid-induced DNA fragmentation, was capable of upregulating NFκB. Compellingly, introduction of an undegradable form of the constitutive NFκB inhibitor, IκB, caused DNA fragmentation in several patient isolates, some of which were resistant to glucocorticoid in vitro. Transcription of anti-apoptotic proteins by NFκB was postulated to be responsible for its effects on survival, but Bcl-2 levels did not fluctuate with glucocorticoid or proteasome inhibitor treatment. ^ The in vitro values generated from these studies were organized into a database containing numbers for over 250 patients. Correlation of relevant clinical parameters revealed that levels of spontaneous apoptosis in vitro differ significantly between Rai stages. Importantly, in vitro resistance to nucleoside analogs or glucocorticoids predicted resistance to chemotherapy in vivo, and inability to achieve remission. ^

Measurement of the Inclusive NuMu Charged Current Cross Section on Carbon in the Near Detector of the T2K

T2K has performed the first measurement of nu(mu) inclusive charged current interactions on carbon at neutrino energies of similar to 1 GeV where the measurement is reported as a flux-averaged double differential cross section in muon momentum and angle. The flux is predicted by the beam Monte Carlo and external data, including the results from the NA61/SHINE experiment. The data used for this measurement were taken in 2010 and 2011, with a total of 10.8 x 10(19) protons-on-target. The analysis is performed on 4485 inclusive charged current interaction candidates selected in the most upstream fine-grained scintillator detector of the near detector. The flux-averaged total cross section is (phi) = (6.91 +/- 0.13(stat) +/- 0.84(syst)) x 10(-39) cm(2)/nucleon for a mean neutrino energy of 0.85 GeV.

Evidence of Electron Neutrino Appearance in a Muon Neutrino Beam

The T2K Collaboration reports evidence for electron neutrino appearance at the atmospheric mass splitting, vertical bar Delta m(32)(2)vertical bar approximate to 2.4 X 10(-3) eV(2). An excess of electron neutrino interactions over background is observed from a muon neutrino beam with a peak energy of 0.6 GeV at the Super-Kamiokande (SK) detector 295 km from the beam's origin. Signal and background predictions are constrained by data from near detectors located 280 m from the neutrino production target. We observe 11 electron neutrino candidate events at the SK detector when a background of 3.3 +/- 0.4(syst) events is expected. The background-only hypothesis is rejected with a p value of 0.0009 (3.1 sigma), and a fit assuming nu(mu) -> nu(e) oscillations with sin (2)2 theta(23) = 1, delta(CP) = 0 and vertical bar Delta m(32)(2)vertical bar = 2.4 X 10(-3) eV(2) yields sin (2)2 theta(13) = 0.088(-0.039)(+0.049)(stat + syst).

Limits on Spin-Dependent WIMP-Nucleon Cross Sections from 225 Live Days of XENON100 Data

We present new experimental constraints on the elastic, spin-dependent WIMP-nucleon cross section using recent data from the XENON100 experiment, operated in the Laboratori Nazionali del Gran Sasso in Italy. An analysis of 224.6 live days x 34 kg of exposure acquired during 2011 and 2012 revealed no excess signal due to axial-vector WIMP interactions with Xe-129 and Xe-131 nuclei. This leads to the most stringent upper limits on WIMP-neutron cross sections for WIMP masses above 6 GeV/c(2), with a minimum cross section of 3.5 x 10(-40) cm(2) at a WIMP mass of 45 GeV/c(2), at 90% confidence level.

The mood-stabilizer lithium prevents hippocampal apoptosis and improves spatial memory in experimental meningitis.

Pneumococcal meningitis is associated with high morbidity and mortality rates. Brain damage caused by this disease is characterized by apoptosis in the hippocampal dentate gyrus, a morphological correlate of learning deficits in experimental paradigms. The mood stabilizer lithium has previously been found to attenuate brain damage in ischemic and inflammatory diseases of the brain. An infant rat model of pneumococcal meningitis was used to investigate the neuroprotective and neuroregenerative potential of lithium. To assess an effect on the acute disease, LiCl was administered starting five days prior to intracisternal infection with live Streptococcus pneumoniae. Clinical parameters were recorded, cerebrospinal fluid (CSF) was sampled, and the animals were sacrificed 42 hours after infection to harvest the brain and serum. Cryosections of the brains were stained for Nissl substance to quantify brain injury. Hippocampal gene expression of Bcl-2, Bax, p53, and BDNF was analyzed. Lithium concentrations were measured in serum and CSF. The effect of chronic lithium treatment on spatial memory function and cell survival in the dentate gyrus was evaluated in a Morris water maze and by quantification of BrdU incorporation after LiCl treatment during 3 weeks following infection. In the hippocampus, LiCl significantly reduced apoptosis and gene expression of Bax and p53 while it increased expression of Bcl-2. IL-10, MCP-1, and TNF were significantly increased in animals treated with LiCl compared to NaCl. Chronic LiCl treatment improved spatial memory in infected animals. The mood stabilizer lithium may thus be a therapeutic alternative to attenuate neurofunctional deficits as a result of pneumococcal meningitis.

2D and 3D crystallization of a bacterial homologue of human vitamin C membrane transport proteins

Most organisms are able to synthesize vitamin C whereas humans are not. In order to contribute to the elucidation of the molecular working mechanism of vitamin C transport through biological membranes, we cloned, overexpressed, purified, functionally characterized, and 2D- and 3D-crystallized a bacterial protein (UraDp) with 29% of amino acid sequence identity to the human sodium-dependent vitamin C transporter 1 (SVCT1). Ligand-binding experiments by scintillation proximity assay revealed that uracil is a substrate preferably bound to UraDp. For structural analysis, we report on the production of tubular 2D crystals and present a first projection structure of UraDp from negatively stained tubes. On the other hand the successful growth of UraDp 3D crystals and their crystallographic analysis is described. These 3D crystals, which diffract X-rays to 4.2Å resolution, pave the way towards the high-resolution crystal structure of a bacterial homologue with high amino acid sequence identity to human SVCT1.

Thiazolides promote apoptosis in colorectal tumor cells via MAP kinase-induced Bim and Puma activation

While many anticancer therapies aim to target the death of tumor cells, sophisticated resistance mechanisms in the tumor cells prevent cell death induction. In particular enzymes of the glutathion-S-transferase (GST) family represent a well-known detoxification mechanism, which limit the effect of chemotherapeutic drugs in tumor cells. Specifically, GST of the class P1 (GSTP1-1) is overexpressed in colorectal tumor cells and renders them resistant to various drugs. Thus, GSTP1-1 has become an important therapeutic target. We have recently shown that thiazolides, a novel class of anti-infectious drugs, induce apoptosis in colorectal tumor cells in a GSTP1-1-dependent manner, thereby bypassing this GSTP1-1-mediated drug resistance. In this study we investigated in detail the underlying mechanism of thiazolide-induced apoptosis induction in colorectal tumor cells. Thiazolides induce the activation of p38 and Jun kinase, which is required for thiazolide-induced cell death. Activation of these MAP kinases results in increased expression of the pro-apoptotic Bcl-2 homologs Bim and Puma, which inducibly bind and sequester Mcl-1 and Bcl-xL leading to the induction of the mitochondrial apoptosis pathway. Of interest, while an increase in intracellular glutathione levels resulted in increased resistance to cisplatin, it sensitized colorectal tumor cells to thiazolide-induced apoptosis by promoting increased Jun kinase activation and Bim induction. Thus, thiazolides may represent an interesting novel class of anti-tumor agents by specifically targeting tumor resistance mechanisms, such as GSTP1-1.

Mechanisms of Ad-p53 induced apoptosis in human malignant glioma

Malignant brain tumors are one of the most challenging cancers affecting society today. In a recent survey, an estimated 17,000 annual cases were recorded with a staggering total of 13,300 deaths. A unique degree of heterogeneity typifies glial tumors and presents a challenge for solitary anti-neoplastic treatments. Tumors subsist as heterogeneous masses that progress through dysplasia to astrocytomas, mixed glioma and glioblastoma multiforme. Although traditional therapeutic approaches have provided increments of success, the median survival time remains 12 months. The urgency to improve upon current clinical protocols has encouraged alternative experimental strategies such as p53 adenoviral gene therapy (Ad-p53). This study addresses the efficacy of Ad-p53 for the treatment of glioma. Our model presents a tumor response that is unique among human cancers. Ad-p53 effectively induces apoptosis in mutant p53 expressing cells yet fails to do so in those with wildtype p53. In order to adopt Adp53 as a standard anti-cancer modality, we characterized the role of the tumor suppressor gene p53 in mediating apoptosis. We demonstrate that altering cellular p53 status through the introduction of a dominant negative mutant p53 (175H, 248W, 273H) sensitized cells to Ad-p53. We discovered that wild-type p53 expressing glioma cells retain the apoptotic machinery necessary to accomplish cell death, but have developed mechanisms that interfere with p53 signaling. Earlier studies have not addressed the mechanisms of Ad-p53 apoptosis nor the resistance exhibited by wild-type p53 glioma. To explain the divergent phenotypes, we identified apoptotic pathways activated and effectors of the response. We illustrated that modulation of the death receptor Fas/APO-1 is a principal means of Ad-p53 signaling that is impaired in wild-type p53 glioma. Moreover, the apoptotic response was found to be a multi-faceted process that engaged several caspases, most notably caspases -1, -3 and -8. Lastly, we assessed the ability of anti-apoptotic molecules Bcl-2 and CrmA to inhibit Ad-p53 apoptosis. These studies revealed that Ad-p53 is a powerful tool for inducing apoptosis that can be delayed but not inhibited by anti-apoptotic means. This work is critical for understanding the development of glioma and the phenotypic and genotypic alterations that account for tumor resistance. ^

Emodin, a tyrosine kinase inhibitor, and human cancer

Many human diseases, including cancers, result from aberrations of signal transduction pathways. The recent understanding of the molecular biochemistry of signal transduction in normal and transformed cells enable us to have a better insight about cancer and design new drugs to target this abnormal signaling in the cancer cells. Tyrosine kinase pathway plays a very important role in normal and cancer cells. Enhanced activity of tyrosine kinases has been associated with many human cancer types. Therefore, identifying the type of tyrosine kinases involved in a particular cancer type and blocking these tyrosine kinase pathways may provide a way to treat cancer. Receptor tyrosine kinase expression, namely epidermal growth factor receptor (EGFR) family, was examined in the oral squamous cell carcinoma patients. The expression levels of different members of the EGFR family were found to be significantly associated with shorter patients' survival. Combining EGFR, HER-2/neu, and HER-3 expression can significantly improve the predicting power. The effect of emodin, a tyrosine kinase inhibitor, on these receptors in head and neck squamous cell carcinoma cell lines was examined. Emodin was found to suppress the tyrosine phosphorylation of HER-2/neu and EGF-induced tyrosine phosphorylation of EGFR. Emodin also induced apoptosis and downregulated the expression of anti-apoptotic protein bcl-2 in oral squamous cell carcinoma cells. It is known that tyrosine kinase pathways are involved in estrogen receptor signaling pathway. Therefore, the effects of inhibiting the tyrosine kinase pathway in estrogen receptor-positive breast cancers was studied. Emodin was found to act similarly to antiestrogens, capable of inhibiting estrogen-stimulated growth and DNA synthesis, and the phosphorylation of Rb protein. Interestingly, emodin, and other tyrosine kinase inhibitors, such as RG 13022 and genistein, depleted cellular levels of estrogen receptor protein. Emodin-induced depletion of estrogen receptor was mediated by the proteasome degradation pathway. In summary, we have demonstrated that tyrosine kinase pathways play an important role in oral squamous cell carcinoma and estrogen receptor-positive breast cancer. Targeting the tyrosine kinases by inhibitors, such as emodin, may provide a potential way to treat the cancer patients. ^

The role of the sonic hedgehog signaling pathway in epidermal homeostasis

Non-melanoma skin cancer is the most frequently diagnosed malignancy in the United States of which basal cell carcinoma (BCC) accounts for 65%. It has recently been determined that deregulation of the sonic hedgehog (shh) pathway leads to the development of BCC. Shh, gli-1, gli-2 gli-3, ptc and smo are overexpressed in BCC and overexpression of these genes in the epidermis results in formation of BCC-like tumors. Despite these observations, the mechanisms by which the pathway controls epidermal homeostasis and the development of the malignant phentotype are unknown. This study assessed the role of the shh pathway in epidermal homeostasis through regulation of apoptosis and differentiation. ^ The anti-apoptotic protein, bcl-2 is overexpressed in BCC, however transcriptional regulators of bcl-2 in the epidermis are unknown. Transient transfection of primary keratinocytes with gli-1 resulted in an increase of bcl-2 expression. Database analysis revealed seven candidate gli binding sites on the bcl-2 promoter. Cotransfection of increasing amounts of gli-1 in keratinoycytes resulted in a corresponding dose-dependent increase in bcl-2 promoter luciferase activity. An N-terminal mutant of gli-3 inhibited gli-1 transactivation of the bcl-2 promoter. The region −428 to −420 was found to be important for gli-1 regulation through gel shift, luciferase assays and site-directed mutagenesis. ^ In order to assess the ability of the shh pathway to regulate keratinocyte differentiation, HaCaT keratinocytes overexpressing sonic hedgehog, were grown in organotypic raft culture. Overexpression of shh induced a basal cell phenotype compared to vector control, as evidenced by transmural staining of cytokeratin 14 and altered Ki67 staining. Shh also induced keratinocyte invasion into the underlying collagen. This was associated with increased phosphorylation of EGFR, jnk and raf and increased expression of c-jun, mmp-9 and Ki67. Interestingly, shh overexpression in HaCaTs did not induce the typical downstream effects of shh signaling, suggesting a gli-independent mechanism. Sonic hedgehog's ability to induce an invasive phenotype was found to be dependent on activation of the EGF pathway as inhibition of EGFR activity with AG1478 and c-225 was able to reduce the invasiveness of HaCaT shh keratinocytes, whereas treatment with EGF augmented the invasiveness of the HaCaT shh clones. ^ These studies reveal the importance of the sonic hedgehog pathway in epidermal homeostasis by regulation of apoptosis through bcl-2, and control of keratinocyte differentiation and invasion through activation of the EGF pathway. They further suggest potential mechanisms by which deregulation of the shh pathway may lead to the development of the malignant phenotype. ^

Mechanism of anti-tumor activity of adenovirus type 5 E1A in ovarian cancer

The adenovirus type 5 E1A gene was originally developed as a gene therapy to inhibit tumorigenicity of HER-2-overexpressing cells by transcriptional downregulation of HER-2. Our goal is to improve the overall efficacy of E1A gene therapy. To achieve this goal, we have conducted two preclinical experiments. ^ First, we hypothesized that Bcl-2 overexpressing ovarian cancer is resistant to E1A gene therapy. This hypothesis is based on that the 19 kDa protein product of the adenoviral E1B gene which is homologous to Bcl-2 inhibits E1A-induced apoptosis. Treating high Bcl-2-xpressing cells with E1A in combination with an antisense oligonucleotide to Bcl-2 (Bcl-2-ASO) resulted in a significant decrease in cell viability due to an increased rate of apoptosis relative to cells treated with E1A alone. In an ovarian cancer xenograft model, mice implanted with low HER-2, high Bcl-2 cells, treated with E1A plus Bcl-2-ASO led to prolonged survival. Bcl-2 thus may serve as a predictive molecular marker enabling us to select patients with ovarian cancer who will benefit significantly from E1A gene therapy. ^ Second, we elucidated the molecular mechanism governing the anti-tumor effect of E1A in ovarian cancer to identify a more potent tumor suppressor gene. We identified PEA-15 (phospho-protein enriched in astrocytes) upregulated in E1A transfected low HER-2-expressing OVCAR-3 ovarian cancer cell, which showed decreased cell proliferation. PEA-15 moved ERK from the nucleus to the cytoplasm and inhibited ERK-dependent transcription and proliferation. Using small interfering RNA to knock down PEA-15 expression in OVCAR-3 cells made to constitutively express E1A resulted in accumulation of phosphoERK in the nucleus, an increase in Elk-1 activity, DNA synthesis, and anchorage-independent growth. PEA-15 also independently suppressed colony formation in some breast and ovarian cancer cell lines in which E1A is known to have anti-tumor activity. We conclude that the anti-tumor activity of E1A depends on PEA-15. ^ In summary, (1) Bcl-2 may serve as a predictive molecular marker of E1A gene therapy, allowing us to select patients and improve efficacy of E1A gene therapy. (2) PEA-15 was identified as a component of the molecular mechanism governing the anti-tumor activity of E1A in ovarian cancer, (3) PEA-15 may be developed as a novel therapeutic gene. ^

Ms. germ. fol. 11 - An Herrn Baron von Lütgendorff [Spottgedicht]. Schreckbar und wohlverdientes Peinliches Urtheil, welches an ... Erdlieb Luftballon ... vollzogen worden

Akzessionsnummer: 38/1847

[Ein Mondhof] : Anno 1645 an St. Thomae Tag den 21. Decembris, Nachts oder Umb 8 der kleinen Uhr nachmittag, hat der Monndt, so noch nicht gar vol, oberhalb gegen Nordwesten, ein Bogen gehabt .̤

Beschreibung des Mondhofes, gefolgt von Berichten über früher beobachtete Mondhoefe

Abüldung underschüdlicher wunder Zeichen : 1663, den 10. December am Montag Neyen Callender, in der Chur Säschischen[!] Bergstatt FreyBurg in meyssen ... am Himlischen Fürmament ist von Meniglichen gesechen worden

Text in d. Platte graviert

Warhafftige Abbildung deß Flecken Plurs, in Grawen Bündten gelegen : wie solcher noch in Flor gewesen, Und auch, wie solcher nach seinem schröcklichen Untergang anzusehen

Auf der zweiten Ill. war wohl der Zustand nach dem Bergrutsch dargestellt