Thrombospondin co-localises with TGFβ and IGF-I in the extracellular matrix of human osteoblast-like cells and is modulated by 17β estradiol

Thrombospondin (TSP) is a multifunctional glycoprotein which is synthesised by several cell types including osteoblasts, and incorporated into the extracellular matrix (ECM) of these cells. The function and regulation of TSP in bone is not clear. In this study, using a long term culture model of human osteoblast-like cells, we examined the distribution of TSP in the ECM and its modulation by added estradiol. In this model the osteoblast-like cells form a regular multilayer which continues to increase in depth up to 50 days post confluence. In the ECM of these cultures and in 19-week fetal bone, the bone markers osteocalcin and alkaline phosphatase were diffusely distributed in the matrix. In contrast, labelling for TSP was concentrated, confined to the banded collagen and its immediately adjacent ECM. This pattern of labelling resembled that of the growth factors transforming growth factor-I (TGF), and insulin-like growth factor-I (IGF-I), with which TSP label co-localised. Labelling intensities were comparable between fetal bone and the in vitro material for TSP, TGF and IGF-I. TSP label was present by 10 days post confluence, reached a maximum by 20 days, and declined slowly thereafter, a time course which was similar to that of IGF-I. Incubation of osteoblast-like cell cultures with 17 estradiol resulted in an increase in multilayer depth and a maximal 3-fold increase in TSP labeling at 30 days as well as approximately 2-fold increases for TGF and IGF-I. The dose-response relationship for these responses to estradiol treatment was biphasic with maximal increases at 10^–10 M–10^–11 M of added estradiol. Treatment with 17 estradiol produced labelling intensities that were not significantly different from controls. Studies with other cell types have suggested that TSP may be involved in modulation of growth factor activity. The similarities between TSP, TGF and IGF-I, in terms of their distribution and regulation by 17 estradiol treatment, may indicate a role for TSP in modulating bone cell proliferation and function through interaction with local growth factors.     

NLRP3 inflammasome activation in macrophage cell lines by prion protein fibrils as the source of IL-1β and neuronal toxicity

Prion diseases are fatal transmissible neurodegenerative diseases, characterized by aggregation of the pathological form of prion protein, spongiform degeneration, and neuronal loss, and activation of astrocytes and microglia. Microglia can clear prion plaques, but on the other hand cause neuronal death via release of neurotoxic species. Elevated expression of the proinflammatory cytokine IL-1β has been observed in brains affected by several prion diseases, and IL-1R-deficiency significantly prolonged the onset of the neurodegeneration in mice. We show that microglial cells stimulated by prion protein (PrP) fibrils induced neuronal toxicity. Microglia and macrophages release IL-1β upon stimulation by PrP fibrils, which depends on the NLRP3 inflammasome. Activation of NLRP3 inflammasome by PrP fibrils requires depletion of intracellular K⁺, and requires phagocytosis of PrP fibrils and consecutive lysosome destabilization. Among the well-defined molecular forms of PrP, the strongest NLRP3 activation was observed by fibrils, followed by aggregates, while neither native monomeric nor oligomeric PrP were able to activate the NLRP3 inflammasome. Our results together with previous studies on IL-1R-deficient mice suggest the IL-1 signaling pathway as the perspective target for the therapy of prion disease.     

Inhibition of proliferation by 1-8U in interferon-α-responsive and non-responsive cell lines

Interferon (IFN)-inducible proteins of the 1-8 gene family mediate homotypic adhesion and transduction of antiproliferative signals. Their induction correlates with inhibition of cell growth while they are often repressed in the course of malignant transformation and tumor development. Ras-mediated transformation of mouse mast cells is associated with downregulation of 1-8U expression and interferon-α (IFN-α) treatment reverts the proliferation rate to normal levels together with induction of 1-8U. Conversely, the antiproliferative responses of IFN-α in sensitive human melanoma cells are accompanied by 1-8U induction. Here we provide direct evidence that recombinant expression of 1-8U in human cell lines is sufficient to block cell proliferation. Based on the abundant expression and subcellular localization to the plasma membrane and exosome-like structures, we propose a model capable of explaining the pleiotropic functions of 1-8 family proteins in tumor cells and during normal development. Received 15 January 2003; received after revision 21 March 2003; accepted 25 March 2003 RID="*" ID="*"Corresponding author.     

Interferon-β can induce the production of plasminogen activator by cultured human cancer cells

Summary Three cultured human cell lines, renal cancer cells (ACHN), bladder cancer cells (EJ), and fibroblasts transformed in culture by Co-60 gamma rays (KMST-6), when treated with interferon-, produced 1.5 to 4 times as much plasminogen activator as the untreated control cultures. This enhanced production of PA was inhibited by cycloheximide or actinomycin D.     

Effect of testosterone and 17-β estradiol on limb regeneration in the newt,Notophthalmus viridescens

Summary Gonadectomy, or injections of testosterone or 17- estradiol, had no apparent effect on the rate of regeneration or histological appearance of limb regenerates in the newt,Notophthalmus viridescens. Neither promotion, nor inhibition of limb regeneration was observed.This work was supported by a grant from the National Research Council of Canada to S.R.S.     

Antitumor effect of β-elemene in non-small-cell lung cancer cells is mediated via induction of cell cycle arrest and apoptotic cell death

-Elemene is a novel anticancer drug, which was extracted from the ginger plant. However, the mechanism of action of -elemene in non-small-cell lung cancer (NSCLC) remains unknown. Here we show that -elemene had differential inhibitory effects on cell growth between NSCLC cell lines and lung fibroblast and bronchial epithelial cell lines. In addition, -elemene was found to arrest NSCLC cells at G2-M phase, the arrest being accompanied by decreases in the levels of cyclin B1 and phospho-Cdc2 (Thr-161) and increases in the levels of p27^kip1 and phospho-Cdc2 (Tyr-15). Moreover, -elemene reduced the expression of Cdc25C, which dephosphorylates/activates Cdc2, but enhanced the expression of the checkpoint kinase, Chk2, which phosphorylates/ inactivates Cdc25C. These findings suggest that the effect of -elemene on G2-M arrest in NSCLC cells is mediated partly by a Chk2-dependent mechanism. We also demonstrate that -elemene triggered apoptosis in NSCLC cells. Our results clearly show that -elemene induced caspase-3, –7 and –9 activities, decreased Bcl-2 expression, caused cytochrome c release and increased the levels of cleaved caspase-9 and poly(ADP-ribose) polymerase in NSCLC cells. These data indicate that the effect of -elemene on lung cancer cell death may be through a mitochondrial release of the cytochrome c-mediated apoptotic pathway.Received 12 January 2005; accepted 5 February 2005     

Interferon-alpha and transforming growth factor-β co-induce growth inhibition of human tumor cells

A hallmark of resistance to type I interferons (IFNs) is the lack of antiproliferative responses. We show here that costimulation with IFN-alpha and transforming growth factor beta-1 (TGF-beta) potentiates antiproliferative activity in a sensitive (ME15) and resistant (D10) human melanoma cell line. A DNA microarray-based search for proliferation control genes involved that are cooperatively activated by IFN-alpha and TGF-beta, yielded 28 genes. Among these are the insulin-like growth factor-binding protein 3 (IGFBP3) and the calcium-binding protein S100A2; we demonstrate, that recombinant IGFBP3 protein is a potent growth inhibitor requiring TGF-beta activity. The antiproliferative activity of S100A2 is significantly enhanced by IFN-alpha in stably transfected ME15 or D10 cell lines. We show for the first time that IFN-alpha is a potent inducer of intracellular calcium release required for activation of S100A2. Our study provides a functional link between IFN-alpha and TGF-beta signaling and extends the function of IFN signaling to calcium-sensitive processes.     

The induction of chromosome aberrations in human lymphocytes by negative π-mesons under conditions of anoxia and oxygenation

Summary After a dose of 3.0 Gy in the peak position of the pion depth-dose curve, the ratios between observed chromosome aberration yields under conditions of oxygenation and of anoxia were obtained for lymphocytes exposed at 3 depths in a plastic phantom. These ratios were 3.7, 1.9 and 1.3 in the plateau, peak and post-peak positions, respectively, suggesting a corresponding decrease in the oxygen enhancement ratio.Acknowledgments. We wish to thank Dr D.H. Reading, Mr M.A. Hynes and Mr W. Spinks for their assistance at the Rutherford Laboratory's -II beam line. This work was partly supported by Euratom Contract 171-76-1 BIO-UK.     

Suppression of cytophilic antibody (‘arming’ factor) in the sera of patients with prostatic cancer by human seminal plasma

Summary The arming of normal peripheral blood leukocytes (PBL) by cytophilic antibody in the sera of prostatic cancer patients is suppressed by pretreatment of PBL with normal human seminal plasma (HuSP1). Suppression of cytophilic antibody by HuSP1 extends the spectrum of immunologic reactions on which SP1 has an immunosuppressive effect and may provide further insight into the possible role of SP1 in the natural history of prostatic cancer.     

Predominant expression of σ and ε globin genes in human leukemia K-562(S6) variant cell line

Summary In the human leukemia K-562(S6) cell line (a) the accumulation ofa-globin chains is low or absent, (b) -globin gene expression is correlated with expression of -chains and (c) the genes responsible for the terminal cell division are not operated within 8–12 cell cycles, while K-562 (S6) cells are fully induced to erythroid differentiation.This work was supported by grants from the Italian National Research Council (C.N.R.) G. R. is a recipient of a fellowship from the National Committee for Nuclear Energy, Rome. Address for reprint requests: R. G., Centro di Studi Biochimici, Via L. Borsari 46, I-44100 Ferrara.     

Levels ofβ-trace protein and lysozyme in human amniotic fluids

Summary The levels of -trace protein and lysozyme were estimated in amniotic fluids from normal fetuses and from fetuses with neuraltube defects. The values of these proteins in normal amniotic fluids were found to be similar to those detected in fetuses with anencephaly and spina bifida. The levels of lysozyme were shown to be correlated with gestational age.     

Contributions in the domain of cancer research: Review¶Human papillomaviruses and their role in cervical cancer

Human papillomaviruses (HPVs) have been linked to a variety of human diseases, most notably cancer of the cervix, a disease responsible for at least 200,000 deaths per year worldwide. Over 100 different types of HPV have been identified and these can be divided into two groups. Low-risk HPV types are the causative agent of benign warts. High-risk HPV types are associated with cancer. This review focuses on the role of high-risk HPV types in cervical tumorigenesis. Recent work has uncovered new cellular partners for many of the HPV early proteins and thrown light on many of the pathways and processes in which these viral proteins intervene. At the same time, structural and biochemical studies are revealing the molecular details of viral protein function. Several of these new avenues of research have the potential to lead to new approaches to the treatment and prevention of cervical cancer.     

Biosynthesis ofα- andβ-ecdysone by the CrayfishOrconectes limosus in vivo and by its Y-Organs in vitro

Summary - and -ecdysone were synthesized from labelled cholesterol by premolt crayfish in vivo and by their Y-organs in vitro.     

Chemokines in neuron–glial cell interaction and pathogenesis of neuropathic pain

Neuropathic pain resulting from damage or dysfunction of the nervous system is a highly debilitating chronic pain state and is often resistant to currently available treatments. It has become clear that neuroinflammation, mainly mediated by proinflammatory cytokines and chemokines, plays an important role in the establishment and maintenance of neuropathic pain. Chemokines were originally identified as regulators of peripheral immune cell trafficking and were also expressed in neurons and glial cells in the central nervous system. In recent years, accumulating studies have revealed the expression, distribution and function of chemokines in the spinal cord under chronic pain conditions. In this review, we provide evidence showing that several chemokines are upregulated after peripheral nerve injury and contribute to the pathogenesis of neuropathic pain via different forms of neuron–glia interaction in the spinal cord. First, chemokine CX3CL1 is expressed in primary afferents and spinal neurons and induces microglial activation via its microglial receptor CX3CR1 (neuron-to-microglia signaling). Second, CCL2 and CXCL1 are expressed in spinal astrocytes and act on CCR2 and CXCR2 in spinal neurons to increase excitatory synaptic transmission (astrocyte-to-neuron signaling). Third, we recently identified that CXCL13 is highly upregulated in spinal neurons after spinal nerve ligation and induces spinal astrocyte activation via receptor CXCR5 (neuron-to-astrocyte signaling). Strategies that target chemokine-mediated neuron-glia interactions may lead to novel therapies for the treatment of neuropathic pain.     

Crosstalk of protein kinase C ε with Smad2/3 promotes tumor cell proliferation in prostate cancer cells by enhancing aerobic glycolysis

Protein kinase C ε (PKCε) has emerged as an oncogenic protein kinase and plays important roles in cancer cell survival, proliferation, and invasion. It is, however, still unknown whether PKCε affects cell proliferation via glucose metabolism in cancer cells. Here we report a novel function of PKCε that provides growth advantages for cancer cells by enhancing tumor cells glycolysis. We found that either PKCε or Smad2/3 promoted aerobic glycolysis, expression of the glycolytic genes encoding HIF-1α, HKII, PFKP and MCT4, and tumor cell proliferation, while overexpression of PKCε or Smad3 enhanced aerobic glycolysis and cell proliferation in a protein kinase D- or TGF-β-independent manner in PC-3M and DU145 prostate cancer cells. The effects of PKCε silencing were reversed by ectopic expression of Smad3. PKCε or Smad3 ectopic expression-induced increase in cell growth was antagonized by inhibition of lactate transportation. Furthermore, interaction of endogenous PKCε with Smad2/3 was primarily responsible for phosphorylation of Ser213 in the Samd3 linker region, and resulted in Smad3 binding to the promoter of the glycolytic genes, thereby promoting cell proliferation. Forced expression of mutant Smad3 (S213A) attenuated PKCε-stimulated protein overexpression of the glycolytic genes. Thus, our results demonstrate a novel PKCε function that promotes cell growth in prostate cancer cells by increasing aerobic glycolysis through crosstalk between PKCε and Smad2/3.     

Purification of platelet-derived endothelial cell growth inhibitor and its characterization as transforming growth factor-β type 1

In 1986, Brown and Clemmons (Proc. natl Acad. Sci. USA83 (1986) 3321) showed that platelets contain a substance, platelet-derived growth inhibitor (PDGI), that inhibits in vitro endothelial cell replication. Although platelets are rich in transforming grwoth factor (TGF-), PDGI was considered not to be related to TGF-, on the basis of its reported properties (extraction from platelets at neutral pH, binding to heparin-Sepharose). However, we purified PDGI to near homogeneity and showed that on the basis of HPLC retention behavior, in vitro growth inhibitory activities with several cell types, receptor binding, and immunoneutralization of growth inhibitory activity with specific anti-TGF- type 1 antibodies, PDGI is most probably identical with TGF- type 1.     

Efficient cell proliferation and predominant accumulation of ɛ-globin mRNA in human leukemic K562 cells which produce mostly Hb Gower 1

Summary Long-term cultures of K562(S) cells in 50–75 M hemin allow the selection of hemin-resistant K562 cells together with cells which proliferate efficiently while fully induced to express the human embryonic globin genes, as the hemoglobin Gower 1 (₂₂) is the predominant hemoglobin produced. Our experiments demonstrate that these K562 cells accumulate mostly -globin mRNA (-globin mRNA/-globin mRNA=2.9) suggesting that the control of hemoglobin expression is at a pretranslational level.We thank Dr Irene Bozzoni (Centro degli Acidi Nucleici, Università di Roma) for the pXCR7 probe. Address for reprint request: R.G. Centro Studi Biochimici sul Morbo di Cooley, Via Borsari 46, I-44100 Ferrara.     

5β-Reduction of [14C]cholesterol by human feces in vitro: Nonspecific inhibition by sugars

Zusammenfassung Nachweis, dass die 5-Reduktion von [¹⁴C]Cholesterol homogenisierter Fäces durch Milch, Laktose und Galaktose gehemmt wurde, wobei die Hemmung unspezifisch war und auch mit anderen Zuckern erzielt werden konnte.