SummaryOpportunistic infections, such as aspergillosis, are among the most serious complications suffered by immunocompromised patients. Aspergillus fumigatus and other pathogenic fungi synthesize a toxic epipolythiodioxopiperazine metabolite called gliotoxin. Gliotoxin exhibits profound immunosuppressive activity in vivo. It induces apoptosis in thymocytes, splenocytes, and mesenteric lymph node cells and can selectively deplete bone marrow of mature lymphocytes. The molecular mechanism by which gliotoxin exerts these effects remains unknown. Here, we report that nanomolar concentrations of gliotoxin inhibited the activation of transcription factor NF-~B in response to a variety of stimuli in T and B cells. The effect ofgliotoxin was specific because, at the same concentrations, the toxin did not affect activation of the transcription factor NF-AT or of interferon-responsive signal transducers and activators of transcription. Likewise, the activity of the constitutively DNA-binding transcription factors Oct-1 and cyclic AMP response element binding protein (CREB), as well as the activation of protein tyrosine kinases p56 lck and p59 fyn, was not altered by gliotoxin. Very high concentrations of gliotoxin prevented NF-~B DNA binding in vitro. However, in intact cells, inhibition of NF-~13 did not occur at the level of DNA binding; rather, the toxin appeared to prevent degradation of IKB-ci, NF-KB's inhibitory subunit. Our data raise the possibility that the immunosuppression observed during aspergillosis results in part from gliotoxin-mediated NF-KB inhibition.

The transforming protein of Rous sarcoma virus (pp6OV'rC) and its normal cellular homologue (pp6Occ) appear to be protein kinases that phosphorylate tyrosine in a variety of protein substrates. In addition, pp6Ov-tc and pp60)-CrC are themselves phosphorylated on serine and tyrosine. It is likely that these phosphorylations serve to regulate the function(s) of pp60v87c and pp6COr. We The transforming gene (v-src) of Rous sarcoma virus (RSV) encodes a 60,000-dalton protein (pp6V-rC) that is capable ofphosphorylating tyrosine in a variety of protein substrates (1-7).Uninfected vertebrate cells contain a similar protein [pp6Ocrc (8)(9)(10)(11)(12)]. Both pp6Ov-rc and pp60c-*c are phosphorylated on serine and tyrosine; these phosphorylations may regulate the enzymatic activity of the proteins. Phosphorylation of cellular proteins by pp60v-rc presumably plays a major role in the establishment and maintenance of the neoplastic phenotype induced by infection with RSV. It is therefore important to explore all of the means by which the action of this protein might be controlled.We have characterized and compared the sites of tyrosine phosphorylation in pp60v-src and pp60CslC. Phosphorylation of the proteins was carried out in two ways: by metabolic labeling of cells in culture (4) and by phosphotransfer in vitro, using partially purified preparations ofpp60v'rcand pp60CS-rC (13,14).Our strategy exploited the availability ofa predicted amino acid sequence for the viral protein (15). Tryptic peptides containing phosphotyrosine were isolated by high-performance liquid chromatography (HPLC) and then subjected to sequential Edman degradation to locate the phosphorylated residue in the peptide. The results indicate that phosphorylation of tyrosine in pp60VS-C occurs on the same amino acid residue either in vitro or in vivo and an apparently identical site is also phosphorylated in pp60Cl-SC in vitro. By contrast, phosphorylation of pp6Ocsrc in vivo apparently occurs at a different site in the protein (16,17). The significance ofthis difference remains to be elucidated. MATERIALS AND METHODSGeneral Procedures. We have described our procedures for the propagation and isotopic labeling ofcultured cells, the preparation of antisera from rabbits bearing tumors induced by the Schmidt-Ruppin strain of RSV, the immunoprecipitation of virus-specific proteins, partial proteolysis by staphylococcus V8 protease, the assay ofthe protein kinase associated with pp6Osrc, the purification ofpp60v-rc by immunoaffinity chromatography, and the fractionation of proteins by electrophoresis in polyacrylamide gels (4,9,13,14). For metabolic labeling of pp6OV-t, chicken embryo fibroblasts were infected with a virus stock that was generated by transfection with cloned SRA-2 DNA (18). Hydrolysis of Proteins with Trypsin. 32P-Labeled polypeptides were visualized by autoradiography ofdried gels, excised, oxidized with performic acid, and digested with N-tosylphenylalanine chloromethyl ketone-trypsin while still in the gel slice, as described by S...

The transforming gene (src) of avian sarcoma virus (ASV) and adjacent regions of the viral genome have been isolated by molelcular cloning of viral DNA. Their nucleotide sequence encompasses the whole of src and the portion of the gene env that encodes gp 37, one of two glycoproteins found in the viral envelope. Src encodes a single, hydrophobic protein with structural features that conform to previous descriptions descriptions of the gene product (pp60src). It appears that a single viral protein is responsible for both the initiation and maintenance of neoplastic transformation by avian sarcoma virus. Neither src nor its product bear any obvious structural relationship to several other viral oncogenes and their encoded proteins. Src is flanked by a repeated nucleotide sequence that may facilitate frequent deletion of the gene from the viral genome.

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Aberrant signaling caused by mutations in the RAS-RAF-MEK-ERK pathway and its upstream activators critically contributes to human tumor development. Strategies, which aim at inhibiting hyperactive signaling molecules, appear conceptually straight forward, but their translation into clinical practice has been hampered by many setbacks. Understanding structure, function and regulation of this intracellular pathway as well as its crosstalk with other signaling activities in the cell will be essential to ensure reasonable usage of new therapeutic possibilities. This review provides an understanding of this signaling cascade as revealed by genetic and biochemical approaches and discusses the existing or arising possibilities to interfere with unphysiological activation in cancer. Signaling aberrations and signal transduction therapies will be discussed exemplary for two types of hematological neoplasia, acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS). In the future understanding the role of tumor stem cells, both as a source of tumor recurrence and tumor heterogeneity, the signals controlling their fate as well as epigenetic changes in cancer will be the next critical steps to further advance the applicability of these novel therapeutic strategies.

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Transcription of DNA from the RNA genome of avian sarcoma virus by IRNA-directed DNA polymerase in vitro initiates on a primer (tRNATrP) located near the 5'-terminus of the viral genome. One of the major products of transcription is a single-stranded DNA chain complementary to a sequence of 101 nucleotides immediately distal to the site of initiation of DNA synthesis. We have determined the complete nucleotide sequence of this transcribed chain for the Prague strain of avian sarcoma virus, a partial sequence of the transcribed chain for the Bratislava 77 strain of avian sarcoma virus, and the sequence of a DNA transcript that is shorter than the transcribed singlestranded chain. Our data define the location of tRNATrP on the genome of avian sarcoma virus and provide the sequence of 119 nucleotides at the 5'-terminus of the genome. Portions of this sequence may be involved in the binding of RNA-directed DNA polymerase, the initiation of translation from viral messenger RNA, the extension of RNA-directed DNA synthesis from the 5'-to the 3'-terminus of viral RNA, and the integration of viral DNA into the host genome.Avian sarcoma viruses (ASV) are retroviruses (1) whose replication requires transcription of DNA from the viral genome by RNA-directed DNA polymerase (2). Transcription from the genome of ASV in vitro initiates on the primer tRNATrP (3, 4) located near the 5'-terminus of the viral RNA (5, 6). One of the major products of transcription is a DNA chain (denoted cDNA,5) complementary to 101 nucleotides of the viral RNA immediately distal to the site of initiation (7-9). We have previously purified and characterized cDNA5' for ASV and we have described the utility of this DNA for the analysis of viral structure and replication (10). We report here the complete sequence of the 101 nucleotides which constitute cDNA5' synthesized with the Prague subgroup C strain (Pr-C) of ASV and a partial sequence for cDNA5' of Bratislava 77 subgroup C strain (B77-C) of ASV.Using the nucleotide sequence of cDNA5' and previous data from our laboratory, we have deduced the sequence of 119 nucleotides at the 5'-terminus of the Pr-C ASV genome. The details of this sequence and its secondary structure are of interest because the 5'-terminus of the viral genome is involved in the initiation of DNA synthesis (7,11,12), the binding of ribosomes for the initiation of viral protein synthesis (13,14), the extension of RNA-directed DNA synthesis from the 5'-terminus to the 3'-terminus of the viral genome (7,11,12), and the integration of viral DNA into the host genome (7,11,12 Cells and Viruses. We have described previously the propagation and purification of ASV (17). B77-C ASV was obtained from R. Friis. Pr-C ASV was provided as concentrated suspensions by University Laboratories through the auspices of the Office of Program Resources and Logistics, National Cancer Institute. Both strains of virus have been extensively propagated and cannot be considered clonal stocks.Isolation of cDNA51. The synthesis and purification of cDNAs...

A large number of G-protein coupled receptors are known to modulate adenylyl cyclase activity. In order to find new compounds modulating the activity of specific receptor subtypes we developed a cellular screening system that measures the biological activity of drugs acting on receptors rather than merely their binding characteristics. The activity of the receptor coupling to the cAMP signal transduction pathway was measured via transcriptional activation of a reporter gene. A chinese hamster ovary cell line was stably transformed with a reporter plasmid containing the firefly luciferase gene under the transcriptional control of multiple cAMP responsive elements (CRE). This CRE reporter cell line exhibited 20 to 30-fold induction of luciferase activity upon stimulation of adenylyl cyclase with forskolin, but did not respond to dopamine agonists. Stable test cell lines were developed by transfecting reporter cell lines with human dopamine D1 and D5 receptor genes, respectively. Treatment of these test cell lines with dopamine receptor agonists and antagonists modulated the luciferase expression in a dose-dependent manner. The rank of potency of dopamine receptor agonists and antagonists was in agreement with reported data obtained from binding studies. The non-isotopic assay can be performed in microtiter plate format and is far less work intensive than the determination of adenylyl cyclase activity by direct cAMP measurement. This technology could also be utilized for discovery of new classes of compounds, e.g. allosteric effectors or non competitive ligands.