Adrian Minty

Monoallelically Expressed Gene Related to p53 at 1p36, a Region Frequently Deleted in Neuroblastoma and Other Human Cancers

We describe a gene encoding p73, a protein that shares considerable homology with the tumor suppressor p53. p73 maps to 1p36, a region frequently deleted in neuroblastoma and other tumors and thought to contain multiple tumor suppressor genes. Our analysis of neuroblastoma cell lines with 1p and p73 loss of heterozygosity failed to detect coding sequence mutations in remaining p73 alleles. However, the demonstration that p73 is monoallelically expressed supports the notion that it is a candidate gene in neuroblastoma. p73 also has the potential to activate p53 target genes and to interact with p53. We propose that the disregulation of p73 contributes to tumorigenesis and that p53-related proteins operate in a network of developmental and cell cycle controls.

Covalent Modification of p73α by SUMO-1 TWO-HYBRID SCREENING WITH p73 IDENTIFIES NOVEL SUMO-1-INTERACTING PROTEINS AND A SUMO-1 INTERACTION MOTIF

Two-hybrid screening in yeast with p73α isolated SUMO-1 (small ubiquitin-likemodifier 1), the enzyme responsible for its conjugation, Ubc-9, and a number of novel SUMO-1-interacting proteins, including thymine DNA glycosylase, PM-Scl75, PIASx, PKY, and CHD3/ZFH. A subset of these proteins contain a common motif,hhXSXS/Taaa, where h is a hydrophobic amino acid and a is an acidic amino acid, that is shown to interact with SUMO-1 in the two-hybrid system. We show here that p73α, but not p73β, can be covalently modified by SUMO-1. The major SUMO-1-modified residue in p73α is the C-terminal lysine (Lys627). The sequence surrounding this lysine conforms to a consensus SUMO-1 modification siteb(X)XXhKXE, whereb is a basic amino acid. SUMO-1-modified p73 is more rapidly degraded by the proteasome than unmodified p73, although SUMO-1 modification is not required for p73 degradation. SUMO-1 modification does not affect the transcriptional activity of p73α on an RGC-luciferase reporter gene in SK-N-AS cells. Instead, SUMO-1 modification may alter the subcellular localization of p73, because SUMO-1-modified p73 is preferentially found in detergent-insoluble fractions. Alternatively, it may modulate the interaction of p73 with other proteins that are substrates for SUMO-1 modification or which interact with SUMO-1, such as those identified here.

IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells.

In animal models of asthma, interleukin-13 (IL-13) induces goblet cell metaplasia, eosinophil infiltration of the bronchial mucosa, and bronchial hyperreactivity, but the basis of its effects on airway epithelia remain unknown. Lesions of the epithelial barrier, frequently observed in asthma and other chronic lung inflammatory diseases, are repaired through proliferation, migration, and differentiation of epithelial cells. An inflammatory process may then, therefore, influence epithelial regeneration. We have thus investigated the effect of IL-13 on mucociliary differentiation of human nasal epithelial cells in primary culture. We show that IL-13 alters ciliated cell differentiation and increases the proportion of secretory cells. IL-13 downregulates the actin-binding protein ezrin and other cytoskeletal components. IL-13 also impairs lateral cell contacts and interferes with the apical localization of ezrin seen in differentiated ciliated cells. In addition, an IL-4 antagonistic mutant protein (Y124D), which binds to the IL-4 receptor alpha subunit, a common chain of IL-4 and IL-13 receptors, inhibits IL-13s effects. IL-13 also decreases ciliary beat frequency in a time- and dose-dependent manner. These results suggest that, in human allergic asthmatic responses, IL-13 affects both ciliated and secretory cell differentiation, leading to airway damage and obstruction.

Human interleukin-13 activates the interleukin-4-dependent transcription factor NF-IL4 sharing a DNA binding motif with an interferon-γ-induced nuclear binding factor

The effects of interleukin‐13 (IL‐13) and interleukin‐4 (IL‐4) on cellular functions were shown to be quite similar. We provide evidence that in monocytes as well as in T lymphocytes both IL‐4 and IL‐ 13 activate the same recently identified transcription factor NF‐IL4 which binds to the specific responsive element IL‐4RE. In addition, we show that a nuclear factor activated by interferon‐γ also interacts with the IL‐4RE. It differs from NF‐IL4 in the electrophoretic mobility of the complex with DNA, in its DNA‐binding specificity and in the proteins interacting with the DNA sequence. Sensitivity against various enzyme inhibitors suggests that components of the signal transduction pathway are shared by all three cytokines.

The pattern of actin expression in human fibroblast × mouse muscle heterokaryons suggests that human muscle regulatory factors are produced

The expression of previously dormant human muscle genes encoding two major components of the contractile apparatus was activated in multinucleated heterokaryons formed by the fusion of mouse muscle cells and human fibroblasts. The accumulation of human and mouse alpha-cardiac and alpha-skeletal actin transcripts was compared by Northern blot, slot blot, and S1 nuclease assays. The pattern of human transcript accumulation in heterokaryons was quite distinct from that in the mouse muscle cells that induced it, and strikingly similar in time course and relative amounts to that in human primary muscle cultures. In addition, the usual decline in the level of mouse alpha-cardiac actin transcripts was not observed; instead, after fusion with human fibroblasts the levels increased. Our findings suggest that the activated human nuclei in heterokaryons produce their own muscle regulatory factors that alter the expression of mouse muscle genes and direct the expression of the human muscle phenotype.

Two-level regulation of cardiac actin gene transcription: muscle-specific modulating factors can accumulate before gene activation.

We have previously proposed that the upstream regions of the human cardiac actin gene contain sequences that interact with muscle-specific factors with direct high-level transcription of this gene in differentiated muscle cells. In this study we showed that these factors already accumulate in the dividing myoblasts of the mouse C2C12 cell line before differentiation of the cells. The endogenous cardiac actin gene in the C2C12 line is expressed only at a low level in myoblasts but at a high level when these cells differentiate into multinucleate myotubes. In contrast, human cardiac actin genes stably introduced into C2C12 cells show high-level expression in both myoblasts and myotubes, indicating that the endogenous cardiac actin gene is repressed in myoblasts by a mechanism which does not affect transfected genes. In a second muscle cell line (the rat L8 cell line), the level of expression of transfected cardiac actin genes increases when these cells differentiate into myotubes, paralleling the expression of the endogenous sarcomeric actin genes. We suggest that the level of transcriptional modulating factors is low in L8 myoblasts and increases when these cells differentiate into myotubes. Our results demonstrate that at least two steps are necessary for high-level cardiac actin gene expression: activation of the gene and subsequent modulation of its transcriptional activity. Furthermore, the results indicate that the two regulatory steps can be dissociated and that the factors involved in modulation are distinct from those involved in gene activation.

Stable polarization of peripheral blood T cells towards type 1 or type 2 phenotype after polyclonal activation

Polarization of T lymphocytes towards type 1 (T1) or type 2 (T2) subsets producing a distinct array of cytokines plays a role in several diseases and could be used for therapeutic intervention. Bearing this purpose in mind, we have established suitable in vitro conditions to drive resting polyclonal human T cells towards stable T1 or T2 polarization profiles. Unse lected peripheral lymphocytes from normal donors were primed with soluble anti‐CD3 monoclonal antibody in the presence of selected sets of recombinant (r) human cytokines. Following this priming process the cytokine secretion profiles of the recovered T cells were assayed after restimulation, both at the population and single‐cell levels. A marked shift towards T2 profile, characterized by heightened production of IL‐4, IL‐5 and IL‐13, was obtained after priming in the presence of rIL‐4 alone. Addition of rIL‐2 partially antagonized this effect. In contrast, priming in the presence of rIL‐2 and rIL‐12 induced a shift towards a T1 pattern characterized by increased productions of IFN‐γ and IL‐2. Strikingly, the T2 profile appeared more stable in culture than the T1 profile. We also observed that the CD4+ helper T cell subset was the major producer of T1 and T2 cytokines after restimulation. These results establish in vitro parameters to deliberately and reproducibly activate resting polyclonal T cells towards a defined and persistent cytokine secretion profile. Autologous T cells polarized under these conditions could be passively transferred as a therapeutic approach in diseases thought to result from imbalance between T1 and T2 responses.

Differential effects of recombinant human interleukin-13 on the in vitro growth of human haemopoietic progenitor cells

Summary. Effects of recombinant human interleukin (IL)‐13 on in vitro haemopoiesis from non‐adherent mononuclear cells (NAMC) or highly enriched CD34+ cells of human cord blood (CB) were studied. IL‐13 significantly increased megakaryocyte (MK) colony formation from either NAMC or CD34+ cells cultured in a plasma clot system supplemented with aplastic anaemia serum (AAS) and phytohaemag‐glutinin‐stimulated human peripheral blood leucocyte‐conditioned medium (PHA‐LCM) in a dose‐dependent manner. Experiments using a modified plasma clot culture, in which normal AB serum and various cytokines were added to replace AAS and PHA‐LCM, demonstrated an increased MK colony number in the presence of IL‐13, especially in combination with IL‐3. However, IL‐13 had no stimulatory effect, but rather a slight inhibitory effect in some cases on granulocyte‐macrophage (GM) colony formation in both plasma clot cultures. Furthermore, the growth of GM progenitor cells in a methylcellulose culture system in the presence of IL‐3, GM‐CSF, Epo, G‐CSF or in combination was significantly inhibited by the addition of IL‐13. On the other hand, high concentrations (lOOng/ml) of IL‐13 were needed to cause a slight inhibition on the growth of BFU‐E‐derived colonies under the same methylcellulose culture. These results indicate that IL‐13, alone and synergistically with the effect of IL‐3, promotes MK colony formation, but it inhibits the growth of GM and erythroid progenitor cells in vitro.

Isolation of an IL-13-dependent subclone of the B9 cell line useful for the estimation of human IL-13 bioactivity

A novel sub-clone of the B9 hybridoma cell line (B9-1-3) has been selected by cloning following continuous culture in rhIL-13. This cell line shows an increased sensitivity to both hIL-13 and mIL-4 compared to the parental B9 cell line. The proliferative response to IL-13 can be blocked with an anti-IL-4 receptor monoclonal antibody but not with the soluble IL-4 receptor, suggesting that IL-13- and IL-4-binding receptor subunits are distinct but form part of a common receptor complex. Although the B9-1-3 cell line is still sensitive to picogrammes of IL-6, it can be used to measure IL-13 in the presence of IL-6 by inclusion of excess neutralizing IL-6 antibody. This cell line should thus prove useful both in measuring the IL-13 bioactivity and for the dissection of the molecular nature of the IL-13:IL-4 receptor complex.