p53-dependent non-coding RNA networks in chronic lymphocytic leukemia.

Mutations of the tumor suppressor p53 lead to chemotherapy resistance and a dismal prognosis in chronic lymphocytic leukemia (CLL). Whereas p53 targets are used to identify patient subgroups with impaired p53 function, a comprehensive assessment of non-coding RNA targets of p53 in CLL is missing. We exploited the impaired transcriptional activity of mutant p53 to map out p53 targets in CLL by small RNA sequencing. We describe the landscape of p53-dependent microRNA/non-coding RNA induced in response to DNA damage in CLL. Besides the key p53 target miR-34a, we identify a set of p53-dependent microRNAs (miRNAs; miR-182-5p, miR-7-5p and miR-320c/d). In addition to miRNAs, the long non-coding RNAs (lncRNAs) nuclear enriched abundant transcript 1 (NEAT1) and long intergenic non-coding RNA p21 (lincRNA-p21) are induced in response to DNA damage in the presence of functional p53 but not in CLL with p53 mutation. Induction of NEAT1 and lincRNA-p21 are closely correlated to the induction of cell death after DNA damage. We used isogenic lymphoma cell line models to prove p53 dependence of NEAT1 and lincRNA-p21. The current work describes the p53-dependent miRNome and identifies lncRNAs NEAT1 and lincRNA-p21 as novel elements of the p53-dependent DNA damage response machinery in CLL and lymphoma.

QuickMap: a public tool for large-scale gene therapy vector insertion site mapping and analysis.

Several events of insertional mutagenesis in pre-clinical and clinical gene therapy studies have created intense interest in assessing the genomic insertion profiles of gene therapy vectors. For the construction of such profiles, vector-flanking sequences detected by inverse PCR, linear amplification-mediated-PCR or ligation-mediated-PCR need to be mapped to the host cell's genome and compared to a reference set. Although remarkable progress has been achieved in mapping gene therapy vector insertion sites, public reference sets are lacking, as are the possibilities to quickly detect non-random patterns in experimental data. We developed a tool termed QuickMap, which uniformly maps and analyzes human and murine vector-flanking sequences within seconds (available at www.gtsg.org). Besides information about hits in chromosomes and fragile sites, QuickMap automatically determines insertion frequencies in +/- 250 kb adjacency to genes, cancer genes, pseudogenes, transcription factor and (post-transcriptional) miRNA binding sites, CpG islands and repetitive elements (short interspersed nuclear elements (SINE), long interspersed nuclear elements (LINE), Type II elements and LTR elements). Additionally, all experimental frequencies are compared with the data obtained from a reference set, containing 1 000 000 random integrations ('random set'). Thus, for the first time a tool allowing high-throughput profiling of gene therapy vector insertion sites is available. It provides a basis for large-scale insertion site analyses, which is now urgently needed to discover novel gene therapy vectors with 'safe' insertion profiles.

New bioinformatic strategies to rapidly characterize retroviral integration sites of gene therapy vectors.

OBJECTIVE: Increasing use of retroviral vector-mediated gene transfer created intense interest to characterize vector integrations on the genomic level. Techniques to determine insertion sites, mainly based on time-consuming manual data processing, are commonly applied. Since a high variability in processing methods hampers further data comparison, there is an urgent need to systematically process the data arising from such analysis. METHODS: To allow large-scale and standardized comparison of insertion sites of viral vectors we developed two programs, IntegrationSeq and IntegrationMap. IntegrationSeq can trim sequences, and valid integration sequences get further processed with IntegrationMap for automatic genomic mapping. IntegrationMap retrieves detailed information about whether integrations are located in or close to genes, the name of the gene, the exact localization in the transcriptional units, and further parameters like the distance from the transcription start site to the integration. RESULTS: We validated the method using 259 files originating from integration site analysis (LM-PCR). Sequences processed by IntegrationSeq led to an increased yield of valid integration sequence detection, which were shown to be more sensitive than conventional analysis and 15 times faster, while the specificities are equal. Output files generated by IntegrationMap were found to be 99.8% identical with results retrieved by much slower conventional mapping with the ENSEMBL alignment tool. CONCLUSION: Using IntegrationSeq and IntegrationMap, a validated, fast and standardized high-throughput analysis of insertion sites can be achieved for the first time.

ProtSweep, 2Dsweep and DomainSweep: protein analysis suite at DKFZ.

The wealth of transcript information that has been made publicly available in recent years has led to large pools of individual web sites offering access to bioinformatics software. However, finding out which services exist, what they can or cannot do, how to use them and how to feed results from one service to the next one in the right format can be very time and resource consuming, especially for non-experts. Automating this task, we present a suite of protein annotation pipelines (tasks) developed at the German Cancer Research Centre (DKFZ) oriented to protein annotation by homology (ProtSweep), by domain analysis (DomainSweep), and by secondary structure elements (2Dsweep). The aim of these tasks is to perform an exhaustive structural and functional analysis employing a wide variety of methods in combination with the most updated public databases. The three servers are available for academic users at the HUSAR open server http://genius.embnet.dkfz-heidelberg.de/menu/biounit/open-husar/

Microarray analysis of newly synthesized RNA in cells and animals.

Current methods to analyze gene expression measure steady-state levels of mRNA. To specifically analyze mRNA transcription, we have developed a technique that can be applied in vivo in intact cells and animals. Our method makes use of the cellular pyrimidine salvage pathway and is based on affinity-chromatographic isolation of thiolated mRNA. When combined with data on mRNA steady-state levels, this method is able to assess the relative contributions of mRNA synthesis and degradation/stabilization. It overcomes limitations associated with currently available methods such as mechanistic intervention that disrupts cellular physiology, or the inability to apply the techniques in vivo. Our method was first tested in serum response of cultured fibroblast cells and then applied to the study of renal ischemia reperfusion injury, demonstrating its applicability for whole organs in vivo. Combined with data on mRNA steady-state levels, this method provided a detailed analysis of regulatory mechanisms of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level.

Retroviral vector insertions in T-lymphocytes used for suicide gene therapy occur in gene groups with specific molecular functions.

Graft-versus-host disease (GvHD) is a severe complication in the context of allogeneic stem cell transplantation and adoptive immunotherapy. The transfer of a suicide gene into donor T-lymphocytes (TLCs) allows selective elimination of GvHD-causing cells. As retroviral gene transfer into hematopoietic stem cells can induce leukaemia, there is an urgent need also to analyze retroviral integration sites in TLCs. We examined suicide gene-transduced TLCs in four grafts and from four transplanted patients. One-hundred and fifteen integration sites were detected in vitro. Of these 90 could be mapped to the human genome; 50% (45) were located in genes and 32% (29) were detected 10 kb upstream or downstream of transcription start sites. We found a significant overrepresentation of genes encoding for proteins with receptor activity, signal transducer activity, transcription regulator activity, nucleic acid binding activity and translation regulator activity. Similar data were obtained from patient samples. Our results point to preferred vector integration patterns, which are specific for the target cell population and probably independent of selection processes. Thus, future preclinical analysis of the integration repertoire with abundant amounts of transduced cells could allow a prediction also for the in vivo situation, where target cells are scarce.

Intracellular distribution of mammalian protein kinase A catalytic subunit altered by conserved Asn2 deamidation.

The catalytic (C) subunit of protein kinase A functions both in the cytoplasm and the nucleus. A major charge variant representing about one third of the enzyme in striated muscle results from deamidation in vivo of the Asn2 residue at the conserved NH(2)-terminal sequence myrGly-Asn-Ala (Jedrzejewski, P.T., A. Girod, A. Tholey, N. König, S. Thullner, V. Kinzel, and D. Bossemeyer. 1998. Protein Sci. 7:457-469). Because of the increase of electronegativity by generation of Asp2, it is reminiscent of a myristoyl-electrostatic switch. To compare the intracellular distribution of the enzymes, both forms of porcine or bovine heart enzyme were microinjected into the cytoplasm of mouse NIH 3T3 cells after conjugation with fluorescein, rhodamine, or in unlabeled form. The nuclear/cytoplasmic fluorescence ratio (N/C) was analyzed in the presence of cAMP (in the case of unlabeled enzyme by antibodies). Under all circumstances, the N/C ratio obtained with the encoded Asn2 form was significantly higher than that with the deamidated, Asp2 form; i.e., the Asn2 form reached a larger nuclear concentration than the Asp2 form. Comparable data were obtained with a human cell line. The differential intracellular distribution of both enzyme forms is also reflected by functional data. It correlates with the degree of phosphorylation of the key serine in CREB family transcription factors in the nucleus. Microinjection of myristoylated recombinant bovine Calpha and the Asn2 deletion mutant of it yielded N/C ratios in the same range as encoded native enzymes. Thus, Asn2 seems to serve as a potential site for modulating electronegativity. The data indicate that the NH(2)-terminal domain of the PKA C-subunit contributes to the intracellular distribution of free enzyme, which can be altered by site-specific in vivo deamidation. The model character for other signaling proteins starting with myrGly-Asn is discussed.

Phosphorylation of the insulin receptor kinase by phosphocreatine in combination with hydrogen peroxide: the structural basis of redox priming.

Signaling by insulin requires autophosphorylation of the insulin receptor kinase (IRK) at Tyr1158, Tyr1162, and Tyr1163. Earlier experiments with (32)P-gamma-ATP indicated that the nonphosphorylated IRK (IRK-0P) is relatively inactive, and crystallographic data indicated that the ATP binding site of IRK-0P is blocked by its activation loop. We now show that phosphocreatine (PCr) in combination with hydrogen peroxide serves as an alternative phosphate donor and that ATP and PCr use distinct binding sites. Whereas phosphorylation of the IRK by ATP is inhibited by the nonhydrolyzable competitor adenylyl-imidodiphosphate, phosphorylation by PCr is enhanced. The IRK mutant Tyr1158Phe showed no phosphorylation with PCr but almost normal phosphorylation with ATP, whereas Tyr1162Phe was phosphorylated well with PCr but less then normal with ATP. 3-Dimensional models of IRK-0P revealed that the conversion of any of the four cysteine residues 1056, 1138, 1234, and 1245 into sulfenic acid produces structural changes that bring Tyr1158 into close contact with Asp1083 and render the well-known catalytic site at Asp1132 and Tyr1162 accessible from a direction that differs from the known ATP binding site. The mutant Cys1138Ala, in contrast, showed relatively inaccessible catalytic sites and weak catalytic activity in functional experiments. Taken together, these findings indicate that 'redox priming' of the IRK facilitates its autophosphorylation by PCr in the activation loop.

Inhibition of the insulin receptor kinase phosphorylation by nitric oxide: functional and structural aspects.

Previous studies on cultured skeletal muscle cells have indicated that the insulin-induced expression of GLUT4 transporter protein is inhibited by nitric oxide (NO). Therefore, we determined the effect of NO on the insulin-induced autophosphorylation of the insulin receptor kinase (IRK), i.e., the first step in the insulin-mediated signal transduction pathway. The experiments showed that the insulin-induced autophosphorylation of the insulin receptor beta-chain is strongly inhibited by the NO donors 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA-NO) or S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect was ameliorated in cells depleted of glutathione (GSH), suggesting the possibility that S-nitroso-glutathione may operate as an intermediate NO donor. Complementary experiments with different Cys --> Ala mutant proteins showed, surprisingly, that all mutant proteins were inhibited by DEA-NO. Three-dimensional models of the nonphosphorylated IR beta-chain nitrosylated at the accessible cysteine residues 1056, 1138, 1234, or 1245 revealed that derivatization of any of these four cysteine residues leads essentially to the same structural changes of the IRK domain. These changes involve a movement of the amino-terminal lobe against the carboxy-terminal lobe in a direction opposite to the direction of the "lobe closure" that was previously proposed to facilitate the accessibility for ATP and the expression of catalytic activity. Our findings suggest that the occurrence of several functionally relevant cysteine residues in distinct regions of the IRK protein increases the probability of regulatory redox interactions and thus the redox sensitivity of the IRK.

W2H: WWW interface to the GCG sequence analysis package.

MOTIVATION: The user-friendly, graphical X-windows interface (WPI) to the GCG sequence analysis package can often not be used due to the lack of an X-server on PC or Macintosh computers. Because Web browsers like Netscape are much more common on those platforms, we decided to develop W2H, a WWW interface to the GCG Sequence Analysis Software Package with nearly the same functionality as the X-windows interface WPI. RESULTS: The new WWW interface (W2H) to the GCG Sequence Analysis Software Package (Wisconsin Package) supports modern Web technologies, like client-pull method, or embedded scripting language, and provides a reasonable platform independence. The interface is quite comprehensive with advanced features like sequence selector, search set builder, enzyme chooser, access to sequence databases, uploading client files to the GCG server or displaying and manipulating graphical outputs in addition to GCG analysis programs. W2H also manages secure access to both GCG server and user data. For special environments, like workshops, conferences and company intranets, there is a special mode (Intranet mode) with less security constraints. The behaviour of W2H is mostly controlled by meta-data files describing the applications and giving a base for dynamic creation of HTML documents. This paper presents mainly the development approaches used, and architectural design aspects of W2H. AVAILABILITY: W2H is available by ftp://ftp.ebi.ac. uk/pub/software/unix/w2h or ftp://genome.dkfz-heidelberg.de/pub/w2h CONTACT: m.senger@ebi.ac.uk

Gap junctional communication and neoplastic transformation.

Gap junctional communication (GJC) is mediated by channels consisting of connexins and can be differentially regulated by ions, second messengers, kinases, phosphatases, and cell adhesion molecules. Tumor cells and oncogene-transformed cells often, but not always, show reduced homologous GJC between themselves. A more stringent correlation may exist between transformation and reduced heterologous communication between transformed cells and normal neighbors. Reduced GJC seems to stimulate tumor promotion but has no significant effect on the initiation phase of carcinogenesis. These effects may reflect the importance of intercellular passage of second messengers or other small molecules in cell growth control. Some evidence suggests that gap junction in combination with cell adhesion molecules can affect metastatic potential, but a clear picture has not yet emerged. Coupling and gap junction expression can be regulated both pre- and posttranslationally in oncogene-transformed cells. Src probably downregulates GJC in fibroblasts by tyrosine phosphorylation of connexin43. The Ras-induced reduction in GJC appears to be caused by decreased connexin expression. E1A, but not Myc and Fos, downregulates GJC to some extent. Artificial expression of connexin in glioma, hepatoma, chemically transformed, and src-transformed cells can restore GJC and suppress growth and/or tumorigenesis. These results argue for involvement of GJC in transformation and growth control.

Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth.

Incorporation of the gene for connexin43, a cell-cell channel protein of gap junction, into the genome of communication-deficient transformed mouse 10T1/2 cells restored junctional communication and inhibited growth. Growth was slowed, saturation density reduced and focus formation suppressed, and these effects were contingent on overexpression of the exogenous gene and the consequent enhancement of communication. In coculture with normal cells the growth of the connexin overexpressors was completely arrested, as these cells established strong communication with the normal ones. Thus, in culture by themselves or in coculture, the connexin overexpressor cells grew like normal cells. These results demonstrate that the cell-cell channel is instrumental in growth control; they are the expected behavior if the channel transmits cytoplasmic growth-regulatory signals.