Donald G. Blair

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.

FLI-1 is a suppressor of erythroid differentiation in human hematopoietic cells.

The FLI-1 oncogene, a member of the ETS family of transcription factors, is associated with both normal and abnormal hematopoietic cell growth and lineage-specific differentiation. We have previously shown that overexpression of FLI-1 in pluripotent human hematopoietic cells leads to the induction of a megakaryocytic phenotype. In this report we show that FLI-1 also acts as an inhibitor of erythroid differentiation. Following the induction of erythroid differentiation, pluripotent cells express reduced levels of FLI-1. In contrast, when FLI-1 is overexpressed in these cells, the levels of erythroid markers are reduced. The ability of FLI-1 overexpressing cells to respond to erythroid-specific inducers such as hemin and Ara-C is also inhibited, and the uninduced cells show a reduced level of the erythroid-associated GATA-1 transcription factor mRNA. Furthermore, expression of a GATA-1 promoter-driven reporter construct in K562 cells is inhibited by co-transfection with a construct expressing FLI-1. Our results support the hypothesis that FLI-1 can act both positively and negatively in the regulation of hematopoietic cell differentiation, and that inhibition of GATA-1 expression may contribute to FLI-1-mediated inhibition of erythroid differentiation.

Cutting edge: Bone morphogenetic protein antagonists Drm/Gremlin and Dan interact with Slits and act as negative regulators of monocyte chemotaxis

Drm/Gremlin and Dan, two homologous secreted antagonists of bone morphogenic proteins, have been shown to regulate early development, tumorigenesis, and renal pathophysiology. In this study, we report that Drm and Dan physically and functionally interact with Slit1 and Slit2 proteins. Drm binding to Slits depends on its glycosylation and is not interfered with by bone morphogenic proteins. Importantly, Drm and Dan function as inhibitors for monocyte migration induced by stromal cell-derived factor 1α (SDF-1α) or fMLP. The inhibition of SDF-1α-induced monocyte chemotaxis by Dan is not due to blocking the binding of SDF-1α to its receptor. Thus, the results identify that Drm and Dan can interact with Slit proteins and act as inhibitors of monocyte chemotaxis, demonstrating a previously unidentified biological role for these proteins.

Ets and retroviruses - transduction and activation of members of the Ets oncogene family in viral oncogenesis.

Studies of retroviral-induced oncogenesis in animal systems led to the initial discovery of viral oncogenes and their cellular homologs, and provided critical insights into their role in the neoplastic process. V-ets, the founding member of the ETS oncogene family, was originally identified as part of the fusion oncogene encoded by the avian acute leukemia virus E26 and subsequent analysis of virus induced leukemias led to the initial isolation of two other members of the ETS gene family. PU.1 was identified as a target of insertional activation in the majority of tumors induced by the murine Spleen Focus Forming virus (SFFV), while fli-1 proved to be the target of Friend murine leukemia virus (F-MuLV) in F-MuLV induced erythroleukemia, as well as that of the 10A1 and Graffi viruses. The common features of the erythroid and myeloid diseases induced by these viruses provided the initial demonstration that these and other members of the ETS family play important roles in hematopoietic development as well as disease. This review provides an overview of the role of ETS genes in retrovirally induced neoplasia, their possible mechanisms of action, and how these viral studies relate to current knowledge of the functions of these genes in hematopoiesis.

Oncogenicity of DNA in vivo: tumor induction with expression plasmids for activated H-ras and c-myc.

All vaccines and other biological products contain contaminating residual DNA derived from the production cell substrate. Whether this residual cell-substrate DNA can induce tumors in vaccine recipients and thus represent a risk factor has been debated for over 50 years without resolution. As a first step in resolving this issue, we have generated expression plasmids for the activated human H-ras oncogene and for the murine c-myc proto-oncogene. Their oncogenic activity was confirmed in vitro using the focus-formation transformation assay. Two strains of adult and newborn immune-competent mice were inoculated with different amounts of either plasmid alone or with a combination of the H-ras and c-myc plasmids. Tumors developed only in mice inoculated with both plasmids and only at the highest amount of DNA (12.5 microg of each plasmid). The NIH Swiss mouse was more sensitive than the C57BL/6 mouse, and newborn animals were more sensitive than adults. Cell lines were established from the tumors. PCR and Southern hybridization analyses demonstrated that both inoculated oncogenes were present in all of the tumor-derived cell lines and that the cells in the tumors were clonal. Western analysis demonstrated that both oncoproteins were expressed in these cell lines. These results demonstrate that cellular oncogenes can induce tumors following subcutaneous inoculation. Such information provides a possible way of evaluating and estimating the theoretical oncogenic risk posed by residual cell-substrate DNA in vaccines.

Suppression of the Ewing's sarcoma phenotype by FLI1/ERF repressor hybrids.

Fusion of the 5′ half of the Ewing’s sarcoma (ES) gene EWS with the DNA-binding domain of several transcription factors has been detected in many human tumors. The t(11;22)(q24;q12) chromosomal translocation is specifically linked to ES and primitive neuroectodermal tumors and results, in the majority of cases, in the fusion of the amino terminus of the EWS gene to the carboxyl-terminal DNA-binding domain of the FLI1 gene. The chimeric protein has been shown to be oncogenic, a potent transcriptional activator, and necessary for the maintenance of the Ewing’s phenotype, making it an attractive target for gene therapy. In this study, we demonstrate that the ES transformed phenotype can be suppressed by chimeric transcriptional repressors containing the DNA-binding domain of FLI1 and the regulatory and repressor domain of ERF, a transcription suppressor and member of the ets gene family. The hybrid repressor is expressed at levels comparable with EWS/FLI1, does not affect EWS/FLI1 expression, and exhibits similar DNA-binding specificity but suppresses transcriptional activity. The FLI1/ERF repressor, like the wild-type ERF, is regulated by mitogen-activated protein kinase-dependent subcellular localization. Our data suggest that transformation by EWS/FLI1 may partially be due to activation of specific EWS/FLI1-regulated genes involved in cell proliferation.

A MOUSE FIBROBLAST LINE CYCLES BETWEEN MONOLAYER AND SPHEROID FORMS, REGULATES MET AND HGF EXPRESSION, AND RELEASES AN ATTACHMENT AND GROWTH-PROMOTING SUBSTANCE

A subline of mesoderm‐derived mouse NIH3T3 fibroblasts was selected for its ability to proliferate in serum‐free media. This cell line (SFDH) grows as a monolayer at low density and spontaneously forms dense, multicellular spheroids at high density. Spheroid formation can also be induced by the addition of dexamethasone, polybrene, or heparin. Spheroids eventually detach from the substrate, but will reattach and re‐form monolayers when transferred to fresh culture vessels and media, repeating the cycle again upon reaching high density. Thin section analysis of spheroids shows morphologically‐distinct regions of cells, including an attenuated outer surface and a cuboidal interior with occasional lumen‐like areas. Over time in culture, spheroids express increasing levels of met, the Met ligand‐SF/HGF and cytokeratin, an epithelial marker, in comparison to monolayers. Both monolayer and spheroid‐derived cells are rapidly tumorigenic in nude mice. Media conditioned by SFDH cells contain factors that stimulate growth and attachment of a variety of tumorigenic and non‐tumorigenic cell lines, inducing cells to divide in serum‐free media for up to 14 days when plated on tissue culture‐treated and nontreated plastic surfaces pre‐coated with SFDH conditional media. The growth‐stimulating activity fractionates as a single peak over a sepharose column in the presence of 6m urea, and sediments as a high molecular weight complex. Growth‐stimulating activity can be neutralized by several antisera specific for hepatocyte growth factor, and the same sera recognize a novel ∼37kD protein in active supernatants. The cyclic, continuous nature of alternating monolayer and spheroid forms makes this cell line appropriate for studying changing gene expression patterns in progressive cell—cell/cell—matrix interactions.

A Mouse Strain Defective in Both T Cells and NK Cells Has Enhanced Sensitivity to Tumor Induction by Plasmid DNA Expressing Both Activated H-Ras and c-Myc

As part of safety studies to evaluate the risk of residual cellular DNA in vaccines manufactured in tumorigenic cells, we have been developing in vivo assays to detect and quantify the oncogenic activity of DNA. We generated a plasmid expressing both an activated human H-ras gene and murine c-myc gene and showed that 1 µg of this plasmid, pMSV-T24-H-ras/MSV-c-myc, was capable of inducing tumors in newborn NIH Swiss mice. However, to be able to detect the oncogenicity of dominant activated oncogenes in cellular DNA, a more sensitive system was needed. In this paper, we demonstrate that the newborn CD3 epsilon transgenic mouse, which is defective in both T-cell and NK-cell functions, can detect the oncogenic activity of 25 ng of the circular form of pMSV-T24-H-ras/MSV-c-myc. When this plasmid was inoculated as linear DNA, amounts of DNA as low as 800 pg were capable of inducing tumors. Animals were found that had multiple tumors, and these tumors were independent and likely clonal. These results demonstrate that the newborn CD3 epsilon mouse is highly sensitive for the detection of oncogenic activity of DNA. To determine whether it can detect the oncogenic activity of cellular DNA derived from four human tumor-cell lines (HeLa, A549, HT-1080, and CEM), DNA (100 µg) was inoculated into newborn CD3 epsilon mice both in the presence of 1 µg of linear pMSV-T24-H-ras/MSV-c-myc as positive control and in its absence. While tumors were induced in 100% of mice with the positive-control plasmid, no tumors were induced in mice receiving any of the tumor DNAs alone. These results demonstrate that detection of oncogenes in cellular DNA derived from four human tumor-derived cell lines in this mouse system was not possible; the results also show the importance of including a positive-control plasmid to detect inhibitory effects of the cellular DNA.