CDC6 is conserved during evolution and is essential and limiting for the initiation of eukaryotic DNA replication. Human CDC6 activity is regulated by periodic transcription and CDK-regulated subcellular localization. Here, we show that, in addition to being absent from nonproliferating cells, CDC6 is targeted for ubiquitin-mediated proteolysis by the anaphase promoting complex (APC)/cyclosome in G 1 . A combination of point mutations in the destruction box and KEN-box motifs in CDC6 stabilizes the protein in G 1 and in quiescent cells. Furthermore, APC, in association with CDH1, ubiquitinates CDC6 in vitro, and both APC and CDH1 are required and limiting for CDC6 proteolysis in vivo. Although a stable mutant of CDC6 is biologically active, overexpression of this mutant or wild-type CDC6 is not sufficient to induce multiple rounds of DNA replication in the same cell cycle. The APC-CDH1-dependent proteolysis of CDC6 in early G 1 and in quiescent cells suggests that this process is part of a mechanism that ensures the timely licensing of replication origins during G 1 .

RAR and AML1 transcription factors are found in leukemias as fusion proteins with PML and ETO, respectively. Association of PML-RAR and AML1-ETO with the nuclear corepressor (N-CoR)/histone deacetylase (HDAC) complex is required to block hematopoietic differentiation. We show that PML-RAR and AML1-ETO exist in vivo within high molecular weight (HMW) nuclear complexes, reflecting their oligomeric state. Oligomerization requires PML or ETO coiled-coil regions and is responsible for abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors. Fusion of RAR to a heterologous oligomerization domain recapitulated the properties of PML-RAR, indicating that oligomerization per se is sufficient to achieve transforming potential. These results show that oligomerization of a transcription factor, imposing an altered interaction with transcriptional coregulators, represents a novel mechanism of oncogenic activation.

PTX3 is a prototypic long pentraxin consisting of a C-terminal 203-amino acid pentraxin-like domain coupled with an N-terminal 178-amino acid unrelated portion. The present study was designed to characterize the structure and ligand binding properties of human PTX3, in comparison with the classical pentraxins C-reactive protein and serum amyloid P component. Sequencing of Chinese hamster ovary cell-expressed PTX3 revealed that the mature secreted protein starts at residue 18 (Glu). Lectin binding and treatment with N-glycosidase F showed that PTX3 is N-glycosylated, sugars accounting for 5 kDa of the monomer mass (45 kDa). Circular dichroism analysis indicated that the protein consists predominantly of ␤-sheets with a minor ␣-helical component. While in gel filtration the protein is eluted with a molecular mass of Х900 kDa, gel electrophoresis using nondenaturing, nonreducing conditions revealed that PTX3 forms multimers predominantly of 440 kDa apparent molecular mass, corresponding to decamers, and that disulfide bonds are required for multimer formation. The ligand binding properties of PTX3 were then examined. As predicted based on modeling, inductive coupled plasma/atomic emission spectroscopy showed that PTX3 does not have coordinated Ca 2؉ . Unlike the classical pentraxins CRP and SAP, PTX3 did not bind phosphoethanolamine, phosphocholine, or high pyruvate agarose. PTX3 in solution, bound to immobilized C1q, but not C1s, and, reciprocally, C1q bound to immobilized PTX3. Binding of PTX3 to C1q is specific and saturable with a K d 7.4 ؋ 10 ؊8 M as determined by solid phase binding assay. The Chinese hamster ovary cellexpressed pentraxin domain bound C1q when multimerized. Thus, as predicted on the basis of computer modeling, the prototypic long pentraxin PTX3 forms multimers, which differ from those formed by classical pentraxins in terms of protomer composition and requirement for disulfide bonds, and does not recognize CRP/SAP ligands. The capacity to bind C1q, mediated by the pentraxin domain, is consistent with the view that PTX3, produced in tissues by endothelial cells or macrophages in response to interleukin-1 and tumor necrosis factor, may act as a local regulator of innate immunity.

SUMMARYPTX3 is a secreted molecule which consists of a C-terminal domain similar to classical pentraxins (e.g. C-reactive protein (CRP)) and of an unrelated N-terminal domain. Unlike the classical pentraxins, the long pentraxin PTX3 is expressed in response to IL-1b and tumour necrosis factor-alpha (TNF-a), but not to IL-6, in various cell types. The present study was designed to investigate the expression of PTX3 in RA. Dissociated RA and osteoarthritis (OA) type B synoviocytes were cultured in the presence and in the absence of inflammatory cytokines. PTX3 mRNA expression in synoviocytes was evaluated by Northern analysis. PTX3 protein levels in synovial cell cultures and synovial fluid were estimated by ELISA, and PTX3 distribution in synovial tissues by immunohistochemical techniques. OA synoviocytes were induced to express high levels of PTX3 mRNA by TNF-a, but not by other cytokines including IL-1b and IL-6. RA synoviocytes, unlike OA synoviocytes, constitutively expressed high levels of PTX3 in the absence of deliberate stimulation. The constitutive expression of PTX3 in RA synoviocytes was not modified by anti-TNF-a antibodies, IL-1 receptor antagonist or a combination of the two agents. In contrast, interferon-gamma and transforming growth factor-beta inhibited PTX3 constitutive expression in RA synoviocytes. The joint fluid from RA patients contained higher levels of immunoreactive PTX3 than controls and the synovial tissue contained endothelial cells and synoviocytes positive for PTX3 by immunohistochemistry. In conclusion, PTX3 may play a role in inflammatory circuits of RA, and its relevance as a marker of disease activity deserves further study.

The ubiquitin pathway has been implicated in the regulation of the abundance of proteins that control cell growth and proliferation. We have identified and characterized a novel human ubiquitin isopeptidase, UBPY, which both as a recombinant protein and upon immunoprecipitation from cell extracts is able to cleave linear or isopeptide-linked ubiquitin chains. UBPY accumulates upon growth stimulation of starved human fibroblasts, and its levels decrease in response to growth arrest induced by cell-cell contact. Inhibition of UBPY accumulation by antisense plasmid microinjection prevents fibroblasts from entering S-phase in response to serum stimulation. By increasing or decreasing the cellular abundance of UBPY or by overexpressing a catalytic site mutant, we detect substantial changes in the total pattern of protein ubiquitination, which correlate stringently with cell proliferation. Our results suggest that UBPY plays a role in regulating the overall function of the ubiquitin-proteasome pathway. Affecting the function of a specific UBP in vivo could provide novel tools for controlling mammalian cell proliferation.

No abstract

The beta-core fragment of hCG proved to be the most effective part of the hCG molecule, inducing growth inhibition and apoptosis of KS cells. Thus, the beta-core could be the most appropriate hCG derivative for the therapy of KS.

SummaryMolecules representative of different classes of chemotactic agents, including formyl-Met-LeuPhe (FMLP), CSa, leukotriene B4, platelet-activating factor, and interleukin (IL)-8, caused a rapid reduction in the IL-1 binding capacity by human polymorphonuclear leukocytes (PMN), a cell type expressing predominantly the IL-1 type II decoy receptor (IL-1 decoy RII). N-t-BocMet-Leu-Phe, an antagonist for the FMLP receptor, inhibited the loss of IL-1 binding capacity induced by FMLP. Monocyte chemotactic protein 1, a chemokine related to IL-8 but inactive on PMN, had no effect on IL-1 binding in this cell type. FMLP was selected for further detailed analysis of chemoattractant-induced loss of IL-1 binding by PMN. The action of FMLP was rapid, reaching 50% of its maximum (80%) at 30 s, the earliest measurable time point, and plateauing between 10 and 30 min. Dose-response analysis revealed that maximal reduction of IL-1 binding was reached at FMLP concentrations that were also optimal for chemotaxis (50% effective dose = 5 x 10 -9 M). The loss of IL-1 binding capacity caused by FMLP was determined by a reduction in receptor number with no change in their affinity. The effect of FMLP on IL-1 receptor (IL-1R) was selective in that the PMN surface structures IL-SR, CD16, CD18, and major histocompatibility complex class I antigens were unaffected under these conditions. Loss of surface IL-1R was not due to an augumented rate of internalization. FMLP caused rapid release of a 45-kD IL-1-binding molecule identified as the IL-1 decoy RII. After FMLP-induced release, PMN reexpressed newly synthesized receptors, reaching basal levels by 4 h. FMLP-induced release of the IL-1 decoy RII did not impair the responsiveness of PMN to IL-1 in terms of promotion of survival and cytokine production. FMLP-induced release of the IL-1 decoy RII was unaffected by protein synthesis inhibitors, was blocked by certain protease inhibitors, and was mimicked by agents (the Ca + + ionophore A23187 and phorbol myristate acetate) that recapitulate elements in the signal transduction pathway of chemoattractant receptors. The time frame and concentration range of chemoattractant-induced rapid release of the IL-1 decoy RII are consistent with the view that IL-1 decoy RII release is an early event in the multistep process of leukocyte recruitment. Rapid chemoattractant-induced IL-1 decoy RII release in the circulation may counteract IL-1 leaking into the systemic circulation from sites of inflammation while preserving the capacity of leukocytes to respond to IL-1 in tissues. This phenomenon may contribute as well to the antiinflammatory effect of systemic administration of chemotactic agents. (1, 2). IL-1 signaling activity appears to be mediated exclusively via the IL-1RI, whereas the IL-1RII has no signaling property and acts in myelomonocytic cells as a decoy for IL-1, inhibiting its activity by preventing IL-1 from binding to the IL-1RI (3, 4).A soluble version of IL-1 decoy RII has been identified in the supernatants from B lyrnphoblastoid cel...