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Results for “Down-Regulation”

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PubMedDate not supplied

Biological roles of HIV Nef proteins. A minireview.

The nef gene, its protein products and diverse mechanisms by which HIV pathogenicity is nef-mediated in vivo and in vitro explain the huge amount of works on this topic. Until now the following functional roles have been assigned for nef: 1. downregulation of virus replication; 2. GTP binding and GTPase activities; 3. modulation of cytoplasmic signalling; and 4. cellular (CD4 and IL-2) gene regulation. Many reports which demonstrate the possible functions of nef in viral replication and in development of AIDS have been refuted by other scientists who failed to confirm some biological activities. Host immune response against nef proteins has been claimed as an early diagnosis marker or to be involved in disease progression. Also, nef proteins have been involved in blocking of HLA antigens, in superantigen production or in crossreactivity with some cellular antigens. The role of nef is a complex one, important in establishing and maintaining viral latency in vivo and regulating virus replication in vitro.

Down-Regulation↗
PubMed1992

Downregulation of saturated fatty acid and upregulation of unsaturated fatty acid by 13-cis-retinoic acid in human prostate cancer cells.

Retinoids play a major role in regulation of epithelial cell growth and cellular differentiation, but their mechanism(s) of action are still unclear. In the present study, we examined the effects of 13-cis-retinoic acid (13-cis-RA) on cytotoxicity, growth properties, morphology, neutral lipids, phospholipids and fatty acids in cultured human prostate cancer cell lines. The results of these experiments suggest that 13-cis-RA (10 microM) inhibits the DNA synthesis and nude mice tumorigenicity by 2- to 3-fold, compared to control. Electron microscopy revealed more differentiated phenotypes after 13-cis-RA treatment. There was a significant increase in phosphatidylcholine and decrease in sphingomyelin in 13-cis-RA treated cells compared to control. The saturated fatty acids significantly decreased whereas unsaturated fatty acids were increased after 13-cis-RA treatment in prostate cancer cells. This study demonstrates for the first time that retinoic acid mediated downregulation of saturated fatty acids and upregulation of unsaturated fatty acid in human prostate cancer cells.

Animals↗
PubMed1992

Tissue distribution of MHC class II--positive cells, their down-manipulation by monoclonal antibodies and potential role in organ allograft immunogenicity.

This review article presents current state of knowledge of tissue distribution of MHC class II antigens in rat kidney. Our attention focuses particularly on down manipulation of these antigens by monoclonal antibodies. The results of organ perfusion procedure on prolongation of graft survival and perspectives of further studies are also discussed.

Animals↗
PubMed1992

The role of Ca2+ in cadmium-induced renal tubular cell injury.

Changes in rat renal proximal tubule cell gene expression were studied in vitro using a cadmium metallothionein (CdMT) exposure model. Increased expression of MT together with alterations in the expression of several other stress protein products were demonstrated by 35S-two-dimensional gel electrophoresis. Stress proteins with estimated molecular masses of 32, 70 and 90 kDa were observed to be increased after 4 h of exposure. These changes preceded the onset of cell injury. Internal stores of Ca2+ were not found to be measurably altered, as measured by the changes in fluorescence of the dye FURA-2. These data suggest that the observed alterations in gene expression are directly mediated by Cd2+ and not secondary to increased availability of Ca2+ within the target cell population, perhaps mediated by the transacting factor which regulates MT synthesis. The calcuria resulting from in vivo exposure to Cd2+ in workers or CdMT exposure in animal models may be secondary to alterations in renal proximal tubule cell gene expression.

Animals↗
PubMed1992

Neuropeptide signal transduction in lung cancer: clinical implications of bradykinin sensitivity and overall heterogeneity.

To define the role of neuropeptides in lung cancer biology, we evaluated the effect of seven peptide classes on signal transduction and growth in human lung and breast cancer cell lines. Flow cytometric methods were used to quantitate the calcium response in individual cells produced by these peptides alone or in combination. The effects on growth were assessed by [3H]thymidine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and soft agarose colony assays. All lung cancer cells demonstrated calcium responses to one or more peptides with classic small cell lines displaying the greatest responsiveness, followed by variant small cell lines and non-small cell lines. Breast cancer cell lines demonstrated little or no response. There was great variability in the magnitude of calcium response and pattern of response between lung cancer cell lines to individual neuropeptides. Bradykinin was the most potent peptide and produced responses in the highest fraction of lung cancer cell lines. Combinations of peptides produced greater intracellular calcium release than the single peptides, although in less than a quantitatively additive manner. Each peptide produced a refractory period which was peptide class specific. The growth stimulating effects of these neuropeptides were absent or small in magnitude and did not correlate with calcium signal transduction. These results imply that lung cancer cells display a wide sensitivity to neuropeptides but in a very heterogeneous manner. Knowledge of this heterogeneity should be incorporated into the design of antitumor strategies based on this autocrine pathway.

Bombesin↗
PubMed1992

Virus-induced loss of class I MHC antigens from the surface of cells infected with myxoma virus and malignant rabbit fibroma virus.

Shope fibroma virus (SFV) is a leporipoxvirus that causes localized benign fibromas in immunocompetent adult rabbits that spontaneously regress due, in part, to a cell-mediated immune response. Myxoma virus (MYX) and malignant rabbit fibroma virus (MRV) are related leporipoxviruses that induce rapidly lethal generalized infections accompanied by tumors and immunosuppression. Because only these latter two viruses are known to compromise cell-mediated antiviral responses, cell surface levels of class I MHC molecules in SFV-, MRV-, and MYX-infected cells were investigated by fluorescent activated cell sorting analysis using a variety of different anti-HLA mAb. After infection with MYX or MRV there is a rapid decrease in the levels of detectable surface class I epitopes as detected by each antibody and by 24 h postinfection class I MHC Ag levels at the cell surface approach the level of background fluorescence observed with control antibodies. In contrast, only a moderate class I decrease is seen during infection with either SFV or vaccinia virus, an orthopoxvirus that is neither tumorigenic nor immunosuppressive. Surface class I marker loss induced by MYX and MRV is not simply due to nonspecific inhibition of total cellular protein synthesis by the viruses because class I levels decrease much further than the extent measured by estimating surface marker turnover in the presence of the protein synthesis inhibitor cycloheximide. Thus the loss of cellular surface class I molecules greatly exceeds the drop in level caused by complete blockage of host cell gene expression, and must involve removal or masking of preexisting class I epitopes from the cell surface by MRV/MYX. Cell surface levels of the transferrin receptor are unaffected by MYX and MRV infection, suggesting the observed class I decrease is not a nonspecific effect on total cell surface glycoproteins. Analysis of cells infected with MRV/MYX in the presence of cycloheximide or of cytosine arabinoside, an inhibitor of poxviral DNA replication, indicates that the class I marker loss is mediated in part by one or more viral late gene products. A probable explanation is that MRV/MYX late protein(s) interact with the class I MHC complex to either physically sequester these away from the cell surface and inhibit their recycling or else induce a conformational change that precludes recognition by all class I antibodies tested. In either event, we propose that such a major perturbation of the class I MHC complex would likely downregulate the class I-mediated presentation of viral Ag required to initiate cell-mediated immunity to these viruses.

Animals↗
PubMed1992

Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors.

The role of epidermal growth factor (EGF) receptor autophosphorylation sites in the regulation of receptor functions has been studied using cells transfected with mutant EGF receptors. Simultaneous point mutation of 4 tyrosines (Y1068, Y1086, Y1148, Y1173) to phenylalanine, as well as removal of these sites by truncation of the carboxyl-terminal 123 amino acid residues, resulted in reduced receptor phosphorylation of an in vivo specific substrate phospholipase C-gamma 1 to less than 50% compared to the wild-type receptor. The internalization rate constant Ke was also significantly lower in these mutants (0.15/min) compared to cells transfected with wild-type receptor (0.27/min). Additional mutation of tyrosine 992 to phenylalanine in the truncated receptor mutant (Dc-123F) further decreased the receptor internalization rate to a minimal level (ke = 0.07-0.10/min), equivalent to the ke measured for cells expressing kinase-negative receptor (A721). Moreover, tyrosine kinase activity of the Dc-123F receptor toward phospholipase C-gamma 1, compared to wild-type receptor, was reduced by 90%. Taken together, these results show that EGF receptor lacking five autophosphorylation sites functions similar to a kinase-negative receptor. Mutation of tyrosine residue Y992 alone in the context of full length EGF receptor, however, did not affect receptor internalization or kinase activity toward phospholipase C-gamma 1. These data indicate that tyrosine 992 is critical for substrate phosphorylation and internalization only in the context of the truncated receptor, and that minor autophosphorylation sites, such as Y992, may act as compensatory regulatory sties in the absence of the major EGF receptor autophosphorylation sites.

Animals↗
PubMed1992

IFN-gamma mediates increased glucocorticoid receptor expression in murine macrophages.

Exposure of the murine macrophage cell line, RAW 264.7, to murine rIFN-gamma resulted in a significant increase in the number of glucocorticoid receptors (GcR). A doubling in the number of GcR was observed as early as 24 h after rIFN-gamma treatment, and receptor number was maximal by 36 h after rIFN-gamma treatment and represented approximately a fourfold increase. Scatchard analysis indicated that a twofold increase in GcR affinity was concomitant with the rIFN-gamma-induced increase in GcR number in RAW 264.7 cells. Increased GcR numbers were induced after exposure of RAW 264.7 cells to as little as 0.1 U/ml rIFN-gamma, and optimal expression was observed at 5 U/ml. Treatment of peritoneal exudate macrophages from C3H/OuJ mice and the LPS hyporesponsive mouse strain, C3H/HeJ, with rIFN-gamma induced an approximately twofold increase in the GcR with no concomitant change in receptor affinity. These results suggest that IFN-gamma may be essential not only for macrophage activation, but also for increasing macrophage sensitivity to feedback inhibition by glucocorticoids by increasing the number and/or affinity of available GcR.

Animals↗
PubMed1992

Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver.

Serine palmitoyltransferase, 3-dehydrosphinganine reductase and sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxosphinganine, sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits serine palmitoyltransferase and sphinganine N-acyltransferase activities by 30-70%.

Acyltransferases↗
PubMed1992

Suppression of melanoma cell motility factor receptor expression by retinoic acid.

beta-All-trans-retinoic acid (RA) has been shown to inhibit the growth, enhance the differentiation, and suppress the transformed and metastatic properties of certain human and murine melanoma cells. This study examined the effect of RA on the level of a cell surface receptor (M(r) 78,000) (gp78) for an autocrine motility factor, which has been implicated in invasion and metastasis. Treatment of murine melanoma cell lines S91-C2, B16-F1, and K1735-P with RA (10 microM) for 5 days decreased the level of gp78 by 37, 72, and 92%, respectively, as revealed by immunoblotting with monoclonal antibodies raised against gp78. In contrast, RA had only a limited effect on gp78 levels in melanoma cell clones or variant cell lines that are resistant to the growth-inhibitory effects of RA (S91-C154, B16-F10, and K1735-Cl19). Further studies with K1735-P, the most sensitive cell line with respect to modulation of gp78, showed that the decrease in gp78 level required at least 1 microM RA and 4 to 5 days of treatment. The binding of anti-gp78 antibodies to the surface of intact RA-treated cells and to intracellular gp78 in permeabilized cells was also lower than in untreated cells. Furthermore, RA treatment decreased the induction of cell motility, on colloidal gold-coated glass coverslips, by anti-gp78 antibodies, which mimic the effect of autocrine motility factor. The RA-induced decrease in antibody-enhanced cell motility was similar to the time- and RA concentration-dependent decrease in the amount of gp78, suggesting that the two events are related. These results raise the possibility that the previously reported suppression by RA of tumor cell invasion and metastasis may be related, at least in part, to suppression of cell motility resulting from the decreased level of the autocrine motility factor receptor.

Animals↗
PubMed1992

FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines.

The fms-like tyrosine kinase 4 (FLT4) complementary DNA was cloned from a human HEL erythroleukemia cell library by polymerase chain reaction-amplification. We previously reported a partial sequence of FLT4 and showed that the FLT4 gene maps to chromosomal region 5q33-qter (O. Aprelikova, K. Pajusola, J. Partanen, E. Armstrong, R. Alitalo, S. Bailey, J. McMahon, J. Wasmuth, K. Huebner, and K. Alitalo, Cancer Res., 52: 746-748, 1992). Here we present the full-length sequence of the predicted FLT4 protein. The extracellular domain of FLT4 consists of 7 immunoglobulin-like loops, including 12 potential glycosylation sites. On the basis of structural similarities FLT4 and the previously known FLT1 and kinase insert domain-containing receptor tyrosine kinase/fetal liver kinase 1 (KDR/FLK1) receptors constitute a subfamily of class III tyrosine kinases. FLT4 was expressed as 5.8- and 4.5-kilobase mRNAs which were found to differ in their 3' sequences and to be differentially expressed in the HEL and DAMI leukemia cells. Interestingly, a Wilms' tumor cell line, a retinoblastoma cell line, and a nondifferentiated teratocarcinoma cell line expressed FLT4, whereas differentiated teratocarcinoma cells were negative. Most fetal tissues also expressed the FLT4 mRNA, with spleen, brain intermediate zone, and lung showing the highest levels. In in situ hybridization the FLT4 autoradiographic grains decorated bronchial epithelial cells of fetal lung. No evidence was obtained for the expression of FLT4 in the endothelial cells of blood vessels.

Amino Acid Sequence↗
PubMed1992

Human small cell lung cancer expresses the octamer DNA-binding and nervous system-specific transcription factor N-Oct 3 (brain-2).

Small cell lung cancer (SCLC) cells express several characteristics of neuronal cells, including synthesis of neuropeptides and expression of the respective receptors. Establishment and maintenance of the neuronal phenotype of SCLC may depend on expression of gene transcription factors inherent to the central nervous system. The present study shows the nervous system-specific transcription factor N-Oct 3 (brain-2) to be expressed in all 13 SCLC cell lines investigated. Furthermore, N-Oct 3 (brain-2) was also found in SCLC-derived skin metastasis. In contrast, in extracts and RNA of non-SCLC cell lines and non-SCLC tumor tissues, such as lung squamous, large cell, and adenocarcinoma, expression of N-Oct 3 (brain-2) was not detectable. These data support the concept that SCLC cells derive from the neuroectodermal cell lineage since expression of N-Oct 3 (brain-2) protein is highly abundant at the neural tube stage and in the adult restricted to the neuroectodermal cell lineage.

Carcinoma, Small Cell↗
PubMed1992

The downregulation of growth-associated proteins in motoneurons at the onset of synapse elimination is controlled by muscle activity and IGF1.

Axonal growth during development and regeneration coincides with the expression of growth-associated proteins (GAPs), including GAP-43 and tubulin-alpha 1. Following contact with the target region and synaptogenesis, GAPs are downregulated. However, high levels of GAP-43 are expressed in some neurons in the adult CNS, indicating that its function may not be restricted to axonal growth. To define the type of signals that regulate GAP levels during development, we have determined whether GAP downregulation coincides with a defined phase of synapse development and whether it is controlled by the postsynaptic target. Levels of GAP-43 mRNA in spinal motoneurons and protein at the neuromuscular junction were analyzed in the developing neuromuscular system of chick and rat. In both species, GAP-43 mRNA declined rapidly at a time corresponding to the onset of synapse elimination, and nerve terminal GAP-43 immunoreactivity became undetectable with a delay of 2-3 d. In rat motoneurons, GAP-43 and tubulin-alpha 1 mRNA levels declined with a similar time course, indicating that at least two GAPs are downregulated at the same time. Blockade of neuromuscular transmission with either a pre- or a postsynaptically acting toxin prevented GAP downregulation, indicating that target activity affects GAP mRNA levels in motoneurons. Finally, counteracting the activity-dependent decline of muscle insulin-like growth factors during synapse elimination by local subcutaneous injections prevented motoneuron GAP downregulation, suggesting that these growth factors may be involved in the regulation of motoneuron GAPs by the muscle. These results demonstrate that motoneurons undergo target-sensitive changes in GAPs at the onset of synapse elimination. In addition, these results raise the possibility that termination of a presynaptic growth mode may be a prerequisite for synapse elimination.

Animals↗
PubMed1992

Regulation of the 5-hydroxytryptamine1B receptor in opossum kidney cells after exposure to agonists.

The density of 5-hydroxytryptamine (5-HT)1B receptors and their coupling to the inhibition of cAMP accumulation were investigated in opossum kidney cells maintained in culture. The density and properties of the receptor were determined using [125I] iodocyanopindolol as the radioligand. The pharmacological specificity of the binding site was consistent with that expected for a 5-HT1B receptor. Serotonin inhibited forskolin-stimulated cAMP accumulation with an EC50 of 4-8 nM. Compounds known to show selectivity at the 5-HT1B receptor, such as trifluoromethyl-phenylpiperazine and CGS-12066B, also inhibited forskolin-stimulated cAMP accumulation, acting as full agonists with efficacies comparable to that of serotonin. Other beta-adrenergic receptor antagonists, including (-)-pindolol and (-)-alprenolol, bound to the receptor with high affinity and acted as partial agonists capable of inhibiting forskolin-stimulated cAMP accumulation. Exposure of cells to 5-HT resulted in a time- and dose-dependent decrease in the density of 5-HT1B receptors that was not accompanied by a change in the Kd of the binding site for [125I] iodocyanopindolol. A maximum decrease of 60% in the number of 5-HT1B receptors was evident after a 16-hr treatment with 1 microM 5-HT. Concomitant with the observed decrease in the density of receptors was a marked increase in the EC50 for 5-HT-mediated inhibition of forskolin-stimulated cAMP accumulation. The EC50 was increased 4-5-fold after a 16-hr exposure to 1 microM 5-HT, and the maximal level of inhibition was markedly decreased. Whereas pretreatment with moderate concentrations of 5-HT (100-300 nM) for 16 hr produced significant decreases in the density of 5-HT1B receptors and increases in the EC50 for inhibition of forskolin-stimulated cAMP formation, there was little change in the maximal level of inhibition that could be attained. Such a combination of changes could be explained by the presence of "spare" 5-HT1B receptors on these cells.

Animals↗
PubMed1992

[Function, molecular structure and gene expression regulation of hepatocyte growth factor and its receptor].

Hepatocyte growth factor (HGF), a potent mitogen of mature hepatocytes in primary culture, is composed of 69 kDa alpha-chain with four kringle domains and 34 kDa beta-chain. HGF is currently thought to be a pleiotropic factor, that stimulates the growth and motility of various epithelial cells, and that inhibits the growth of some carcinoma cells. These cells have a single class of high affinity receptor with a dissociation constant (Kd) of 20-30 pM. The maximum number of binding sites (Bmax) was determined to be 100-1500 sites/cell. After 70% partial hepatectomy and unilateral nephrectomy, HGF activity in serum and their tissues increased markedly and HGF receptors of the injured organ rapidly decreased, but not in other normal organs. These results suggest that HGF acts only in a regenerating organ after injury through the control mechanisms governing HGF receptor.

Animals↗