The G-protein-coupled formylpeptide receptor FPR confers a more invasive phenotype on human glioblastoma cells.

BACKGROUND: The G-protein-coupled formylpeptide receptor (FPR) that mediates chemotaxis of phagocytic leucocytes induced by bacterial and host-derived chemotactic peptides is selectively expressed by highly malignant human gliomas and contributes to tumour growth and angiogenesis. As invasion of surrounding normal tissues is one of the important features of tumour malignancy, we investigated the function of FPR in the invasive behaviour of human glioblastoma cells. METHODS: Cells (FPR(+) and FPR(-)) were isolated by single-cell cloning from a human glioblastoma cell line U-87MG. The FPR expression was assayed by flow cytometry and reverse transcription PCR. The function of FPR was investigated by chemotaxis and calcium flux induced by FPR agonist fMLF. Tumour cell motility was assayed by a wound-healing model in vitro. The growth and invasive phenotype were observed with subcutaneous implantation of tumour cells in nude mice. Over-expression of FPR in FPR(-) cells was performed by transfection of a plasmid vector-containing human FPR gene. RESULTS: One of the glioma clones F9 that expressed high level of FPR showed a more 'motile' phenotype in vitro as compared with a clone G3 without FPR expression. Although F9 and G3 clones both formed subcutaneous tumours in nude mice, only F9 tumours invaded surrounding mouse connective tissues. Over-expression of FPR in G3 clone (G3F) increased the cell motility in vitro and the capacity of the cells to form more rapidly growing and invasive tumours in nude mice. We further found that, in addition to supernatant of necrotic tumour cells, foetal calf serum and human serum used in culture media contained FPR agonist activity and increased the motility of FPR-expressing glioblastoma cells. CONCLUSION: The expression of FPR is responsible for increased motility of human glioblastoma cells and their formation of highly invasive tumours.

Genomic masking of nondefective recombinant murine leukemia virus in Moloney virus stocks.

HIX virus cloned from Moloney leukemia virus stocks is a nondefective, leukemogenic, and amphotropic murine oncornavirus with a recombinant-type major glycoprotein. Although Moloney leukemia virus stocks generally contain little or no free amphotropic virus, dilution analysis of several virus stocks and the examination of virus progeny from individual foci revealed that HIX virus is present and functionally coated with ecotropic Moloney virus envelopes. Because most mice have serum factors that inactivate recombinant viruses, masking may represent a general survival mechanism for HIX as well as other analogous recombinant viruses.

Retinyl acetate: effect on cellular content of RNA in epidermis in cell culture in chemically defined medium.

Cell cultures of epidermis from newborn mice were established in chemically defined medium. Additions of retinyl acetate to these cultures caused a significant increase in cellular RNA content. Addition of insulin and hydrocortisone to the cultures potentiated the effect of retinyl acetate on cellular RNA content.

Synthetic CD4 peptide derivatives that inhibit HIV infection and cytopathicity.

Synthetic peptide segments of the CD4 molecule were tested for their ability to inhibit infection of CD4+ cells by the human immunodeficiency virus (HIV) and to inhibit HIV-induced cell fusion. A peptide mixture composed of CD4(76-94), and synthesis side products, blocked HIV-induced cell fusion at a nominal concentration of 125 micromolar. Upon high-performance liquid chromatography, the antisyncytial activity of the peptide mixture was found not in the fraction containing the peptide CD4(76-94) itself, but in a side fraction containing derivatized peptide products generated in the automated synthesis. Derivatized deletion and substitution peptides in the region CD4(76-94) were used to demonstrate sequence specificity, a requirement for benzyl derivatization, and a core seven-residue fragment required for antisyncytial activity. A partially purified S-benzyl-CD4(83-94) peptide mixture inhibited HIV-induced cell fusion at a nominal concentration of less than or equal to 32 micromolar. Derivatized CD4 peptides blocked cell fusion induced by several HIV isolates and by the simian immunodeficiency virus, SIV, and blocked infection in vitro by four HIV-1 isolates with widely variant envelope gene sequences. Purified CD4(83-94) dibenzylated at cysteine 86 and glutamate 87 possessed antisyncytial activity at 125 micromolar. Derivatization may specifically alter the conformation of CD4 holoreceptor peptide fragments, increasing their antiviral efficacy.

Lipoprotein-associated oncornavirus-inactivating factor in the genus Mus: effects on murine leukemia viruses of laboratory and exotic mice.

High titers of oncornavirus-inactivating factor (OIF) were found previously in sera of laboratory mice. OIF is highly active against mouse xenotropic and polytropic envelope recombinant murine leukemia viruses (MuLVs) but not against ecotropic MuLVs. Of the 20 different mouse species or subspecies currently tested, that represent 4 subgenera, no OIF was found in the 3 subgenera more distant to the laboratory mouse. In the subgenus Mus, 7 of the 8 most distant species had no OIF, whereas all the ancestral species and subspecies of the laboratory mouse (Mus musculus musculus, Mus musculus domesticus), including a more distant member (Mus musculus cookii), had ample titers of OIF. A new separation technique was devised so that potential virus-neutralizing immunoglobulins could be separated by electrophoresis from OIF in small-volume serum samples. Active OIF was recovered from serum high-density lipoprotein, from very-low-density lipoprotein, as well as from chylomicron fractions. Murine sarcoma virus pseudotypes were made with several available exotic MuLV types. These pseudotype MuLVs were not susceptible to standard OIF preparations. The sera of exotic mice also had no factor analogous to OIF which would inactivate their own homologous or heterologous exotic MuLVs. It appears that, with one exception, OIF activity is limited to two subspecies of M. musculus and may be correlated in these subspecies with the presence of endogenous xenotropic MuLVs.

Amyloid (beta)42 activates a G-protein-coupled chemoattractant receptor, FPR-like-1.

Amyloid beta (Abeta) is a major contributor to the pathogenesis of Alzheimer's disease (AD). Although Abeta has been reported to be directly neurotoxic, it also causes indirect neuronal damage by activating mononuclear phagocytes (microglia) that accumulate in and around senile plaques. In this study, we show that the 42 amino acid form of beta amyloid peptide, Abeta(42), is a chemotactic agonist for a seven-transmembrane, G-protein-coupled receptor named FPR-Like-1 (FPRL1), which is expressed on human mononuclear phagocytes. Moreover, FPRL1 is expressed at high levels by inflammatory cells infiltrating senile plaques in brain tissues from AD patients. Thus, FPRL1 may mediate inflammation seen in AD and is a potential target for developing therapeutic agents.

Synthetic peptide MMK-1 is a highly specific chemotactic agonist for leukocyte FPRL1.

Human phagocytic leukocytes express the seven-transmembrane G-protein-coupled receptors formyl peptide receptor (FPR) and FPR-like 1 (FPRL1). MMK-1, a synthetic peptide derived from a random peptide library, is reported to induce calcium mobilization specifically in human FPRL1 gene-transfected cells. However, its actions on human phagocytic leukocytes remain poorly defined. We found that MMK-1 is a potent chemotactic and calcium-mobilizing agonist for human monocytes, neutrophils, and FPRL1-transfected human embryonic kidney (HEK) 293 cells but is inactive in cells transfected with FPR. MMK-1 also activated HEK 293 cells transfected with FPR2, a mouse counterpart of human FPRL1. Furthermore, MMK-1 increased pertussis toxin-sensitive production of inflammatory cytokines in human monocytes. MMK-1 signaling in human phagocytes was completely desensitized by a well-defined FPRL1 agonist, suggesting that FPRL1 is likely a receptor that mediates the action of MMK-1 in primary cells. Since MMK-1 is one of the most potent FPRL1-specific agonists identified so far, it can serve as a modulator of the host defense and a useful agent for further studying the signaling and function of FPRL1.

Pathogenesis and virus content of lymphomas induced by pure ecotropic Graffi murine leukemia virus.

Tumors induced by wild type Graffi murine leukemia virus (Gi-MuLV) contained high titers of MuLV consisting of a predominant ecotropic (e)-MuLV and a scarcer titer recombinant (RM) MuLV component. Each of these was purified by biological cloning and examined for its envelope properties and leukemogenicity. Both the e- and the RM-MuLV's were single isolates and unique in terms of their neutralization profiles and peptide maps. The cloned e-Gi-MuLV was highly leukemogenic in C57Bl mice, inducing a very rapid lethal thymic lymphoma but no myeloid leukemia. e-Gi-MuLV also accelerated thymic lymphoma in AKR mice. The purified RM-MuLV did not induce any tumors. Infectious cell center (ICC) experiments of organs of mice inoculated with e-Gi-MuLV showed that virus replicated very rapidly and reached maximal titers in about one week in C57Bl mice. There was a highly preferential replication in the thymus of the animal so that this e-Gi-MuLV can be considered as thymotropic. Within two weeks after infection of mice, infected cells of the thymus also began to release low levels of a non-ecotropic MuLV. The rapid induction of lymphoma is compared to that induced by other e-MuLV's and their RM-MuLV's, and to the natural AKR-MuLV-associated disease. These findings are discussed in the context of prevailing theories on envelope gene rearrangements in the virus and in the proviral sequences in resulting tumors.

Expression of functional formyl peptide receptors by human astrocytoma cell lines.

Activation of astrocytes is important in the pathogenesis of a variety of diseases in the central nervous system, such as infection and neurodegeneration. We found that the bacterial chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLF) induced potent migration and Ca(2+) mobilization in human astrocytoma cell lines. The effect of fMLF was pertussis toxin-sensitive, suggesting the involvement of seven transmembrane, G protein-coupled receptor(s) for fMLF. Scatchard analyses revealed that astrocytoma cell lines express both high- and low-affinity binding sites for [3H]fMLF. RT-PCR confirmed the expression of transcripts of fMLF receptors, the high-affinity FPR and the low-affinity FPRL1 by these cells. Both fMLF and F peptide, a synthetic peptide domain of HIV-1 envelope protein which specifically activates FPRL1, increased secretion of IL-6 by astrocytoma cells. Our study demonstrates for the first time that FPR and FPRL1 expressed by astrocytoma cell lines are functional, and suggests a molecular basis for the involvement of these receptors in host defense in the brain.

Simple, rapid, quantitative, syncytium-forming microassay for the detection of human immunodeficiency virus neutralizing antibody.

A simple, rapid, quantitative syncytium-forming microassay for the detection of human immunodeficiency virus (HIV-I) isolates is described. A virus-syncytial sensitive clone of CEM cells (CEM-SS) was identified and made adherent to flat bottom 96-well microtiter dishes. Following the addition of virus, these cells develop easily quantifiable, adherent syncytia on a background of confluent, normal CEM-SS monolayer in 4 to 6 days. One-hit kinetics for syncytia formation were obtained at various multiplicities of infection. Syncytia are associated with complete virion production and cytoplasmic localization of the p24 core protein (detected by immunofluorescence). Total infectious virus can be accurately determined in this assay; these results showed a close correlation with p24 and gp120 induction when microtiter well supernatants were passed to fresh cells and evaluated by competitive radioimmunoassay. Studies of p24 antigen induction at and beyond the end point of syncytia formation indicate that there are no detectable nonsyncytial variants in standard HIV-I stocks. Six divergent HIV-I isolates (HTLV-IIIB, -RFII, -MN, -RUTZ, -CC, and LAV-1), as well as HTLV-IIIB and LAV-1 reisolated from persistently infected chimpanzees, produce quantifiable syncytia which vary slightly in their developmental morphology. Accurate neutralization titers are readily obtained from easily constructed multiplicity curves derived from serial dilutions of test sera. Inherent within this system is a flexible method for studying various kinetics of antibody/virus interactions, as well as blocking and interference studies with any candidate antiviral compounds.

A burgeoning family of biological mediators: chemokines and chemokine receptors.

Chemokines are a superfamily of pro-inflammatory polypeptide cytokines that selectively attract and activate different cell types. Most of its members are small proteins that exhibit conserved cysteines in specific positions. Chemokines activate cells through their binding to shared or unique cell surface receptors which belong to the seven-transmembrane (STM), G-protein-coupled receptors (GPCRs). The large number of chemokines and chemokine receptors are indicative of the importance of these molecules in a variety of pathophysiological conditions.

Identification of virus found in mouse lymphomas induced by HIX murine oncornavirus.

Lymphomas induced by pure HIX murine leukemia virus in two mouse strains contained large amounts of virus with amphotropic properties. Analysis of tumor derived virus purified by limiting dilution techniques indicated that its interference and neutralization spectra were essentially identical to parental HIX virus and different from eco- and xenotropic viruses. Examination of virus progeny from many individual foci induced by virus derived directly from lymphomas indicated that each infectious unit contained only HIX virus. No evidence of ecotropic virus presence was observed.

Selective host range restriction of goat cells for recombinant murine leukemia virus and feline leukemia virus type A.

We isolated a strain of normal goat fibroblasts which was uniquely selective in that it allowed the replication of xenotropic murine leukemia virus but not polytropic recombinant murine leukemia virus. In addition, feline leukemia virus type A replication was severely diminished in these goat cells, whereas feline leukemia virus type B and feline endogenous RD114-CCC viruses replicated efficiently. No other known cells exhibit this pattern of virus growth restriction. These goat cells allow the study of xenotropic murine leukemia virus in mixtures which also contain recombinant murine leukemia virus and may be helpful in eliminating feline leukemia virus type which often coexists in feline sarcoma or leukemia virus mixtures with other feline leukemia virus types.

Generation of recombinant murine leukemia viruses de novo: an alternative model for leukemogenesis.

Evidence for the viral etiology of murine lymphoma is very complex. Although ecotropic viruses were considered in the past to be the causative agents, envelope gene recombinant (RM) types of murine leukemia virus (MuLV) seem to be the best candidates at present. The following factors are relevant in the murine model: Pure ecotropic MuLV causes disease but induces RM-MuLV de novo in every case. RM-MuLV can cause disease in pure form. However, the mere presence of RM-MuLV may not be sufficient to cause disease in some cases. Ecotropic MuLV is needed under natural conditions for inducing T-cell blastogenesis, donating its coat to recombinant MuLV, and to become a partial parent for RM-MuLV. Early elimination of ecotropic MuLV can prevent disease. In a number of virus-free mouse lymphomas, the only indicator of virus involvement was on RM-MuLV glycoprotein on cell surface. Based on the above, a signal hypothesis model is proposed which attempts to integrate the above observations.

Generation of thymotropic envelope gene recombinant virus and induction of lymphoma by ecotropic Moloney murine leukemia virus.

Biologically cloned pure ecotropic Moloney MuLV was used to infect Balb/c and AKR mice to determine the replication of ecotropic virus, the possible generation of recombinant viruses, and the induction of disease. Infectious cell center (ICC) experiments carried out with lymphoid cells of individual Balb/c mice showed that e-M-MuLV rapidly infected up to 30% of lymphoid cells in liver, spleen, and especially in the thymus. No recombinant virus was seen until about Day 35 when a burst of RM-MuLV was observed only in the thymus. New RM-MuLV was found in all 32 preleukemic and leukemic mice tested and persisted at low levels until death. The RM-MuLV recovered early in the preleukemic phase had an env-related M-MuLV but grew very poorly. Cells from a late tumor which grew and cloned readily were examined to see whether the new RM-MuLV was present in every clone. Overtly, most tumor cells did not seem to contain RM-MuLV, but when "unmasking" was performed, every tumor cell contained identical RM-MuLV. In AKR mice, both e-M-MuLV and recombinant M-MuLV caused an acceleration of lymphoma. De novo appearance of a thymotropic RM-MuLV, which was of the Moloney RM-MuLV type and the absence of early detectable endogenous AKR-MCF-type recombinants, suggested that the early lymphoma was due to the induction of a new disease. Several theoretical approaches dealing with viral env-gene permutations are discussed.

A new insight into the role of "old" chemotactic peptide receptors FPR and FPRL1: down-regulation of chemokine receptors CCR5 and CXCR4.

N-formyl peptides, such as fMet-Leu-Phe (fMLP), are some of the first identified and most potent chemoattractants for phagocytic leukocytes. In addition to the bacterial peptide fMLP and the putative endogenously produced formylated peptides, we recently identified a number of other novel peptide agonists that selectively activate the prototype formyl peptide receptor (FPR) and/or its variant FPRL1. These agonists include several synthetic peptide domains derived from the envelope proteins of the human immunodeficiency virus type 1 (HIV-1) and intact human acute phase serum protein serum amyloid A. The activation of FPR and/or FPRL1 in monocytes by these agonists resulted in increased cell migration, calcium mobilisation and the heterologous down-regulation of the expression and function of chemokine receptors, notably CCR5 and CXCR4, two crucial fusion co-receptors for HIV-1. This down-regulation of CCR5 by FPR and FPRL1 agonists was associated with a rapid serine phosphorylation of CCR5. The desensitisation of CCR5 by FPR or FPRL1 agonists, not only inhibited its biological function induced by chemokine ligands, but also interfered with its capacity to act as a fusion co-receptor for monocyte tropic HIV-1. Thus, heterologous desensitisation by FPR and FPRL1 may play an important role in orchestrating the host innate immune responses which generate multiple chemotactic stimulants. Furthermore, the understanding of the structural and biochemical basis of FPR/FPRL1 activation may lead to the development of novel immunoregulatory and anti-HIV agents that emulate the process of heterologous desensitisation.

Relationships between structure and activity of retinoids.

Structure-function relationships of 19 retinoids were studied in three in vitro systems in defined, serum-free medium. These systems measure effects on differentiation and growth, as well as toxic effects. The ring, side chain, and polar terminal group of natural retinoids all may be modified synthetically to yield highly active compounds. Several retinoid ethers were found to be significantly less toxic than the corresponding carboxylic acids or alcohols.

Prevention of chemical carcinogenesis by vitamin A and its synthetic analogs (retinoids).

An approach to chemoprevention of common forms of epithelial cancer, during the period of preneoplasia, is described. Vitamin A and its synthetic analogs (retinoids) are potent agents for control of cell differentiation in many epithelial tissues. Direct effects of retinoids on normal and preneoplastic cell differentiation can be measured in organ culture. In experimental animals, deficiency of dietary retinoids enhances susceptibility to chemical carcinogenesis. Natural retinoids, fed at high dietary levels, have some ability to prevent chemical carcinogenesis in epithelial tissues of bronchi, trachea, stomach, uterus, and skin of experimental animals. However, natural retinoids have limited usefulness for chemoprevention of cancer because of inadequate tissue distribution and excessive toxicity. Synthetic retinoids have been made and shown to be more potent and less toxic for prevention of cancer in animals. Several structural modifications of the ring and terminal portions of the retinoid molecule have significant biological activity; modification of the side chain has been more difficult. The potential future usefulness of this approach to cancer prevention in man will depend on further synthetic modification of the retinoid molecule.