Fischer rat FR3T3 cells were tested for tumorigenicity, invasive and metastatic capabilities before and after transfection, either with the entire bovine papilloma virus type 1 (BPV-1) genome or with a plasmid (pV69) containing a 69 per cent Bam H1-Hind III fragment of the BPV-1 genome as well as bacterial sequences. Cell lines were grouped as parental, pV69-transfectants, BPV-1 transfectants, in vitro derivatives, and in vivo derivatives. The tumorigenic, invasive and metastatic capabilities of these cell lines were examined in vivo through s.c., and i.p. injections of cell suspensions and through s.c. implantations of cellular aggregates into syngeneic rats. Invasiveness was tested in vitro through confrontations with embryonic chick heart fragments in organ culture. All cell lines including parental lines, were found to be invasive in vitro and tumorigenic in vivo; all tumors were invasive. It is, therefore, not possible to draw conclusions about the role of BPV-1 gene sequences in the acquisition of the invasive phenotype. Transfection with BPV-1 genes conveyed the metastatic phenotype upon parental FR3T3 cells, which were themselves found to be non-metastatic. With regards to this, no differences were found between BPV-1 transfectants compared with pV69 transfectants. Untransfected cells became metastatic also through passage in vivo as an s.c. tumor. The expression of the metastatic phenotype was not noticeably correlated with alterations of growth characteristics of the cell lines. We concluded that the implication of BPV-1 gene sequences in conveying the metastatic phenotype upon FR3T3, if any, was indirect, presumably through alterations of the host cell genome. Our experiments illustrate the need for long-term observations with parental cell lines before drawing conclusions about the role of oncogenes in the acquisition of the malignant phenotype.

Animals↗

PubMedDate not supplied

Deletion mapping of the inducible promoter of human IFN-beta gene.

Human fibroblast cells produce beta-type interferon only in response to viral infection or treatment with an inducer such as poly(rI) X poly(rC); this event is most probably controlled at the transcriptional level (for review see ref. 1). To study the induction process, we inserted the human fibroblast interferon (IFN-beta) gene, with or without its promoter region, into recombinant simian virus 40 (SV40) plasmid vectors which subsequently were transfected into monkey AP-8 cells. We report here that upon induction with poly(rI) X poly(rC) there was a 10-30-fold increase in IFN-beta synthesis. This inducer had no effect on interferon production when the coding region only was inserted into the vector plasmid, which indicates that the promoter region is required for inducibility of this gene. Deletion mapping implicates the region between nucleotides -144 and -186 from the mRNA initiation site in the specific regulation of the IFN-beta gene. This region contains a sequence that is remarkably homologous with a consensus sequence found in the 5' flanking region of steroid hormone responsive genes, which might be involved in binding the progesterone-receptor complex.

Animals↗

PubMedDate not supplied

Lymphocytes recognize human vascular endothelial and dermal fibroblast Ia antigens induced by recombinant immune interferon.

T-lymphocyte-mediated responses to the cellular components of blood vessels are important in rejection of allografts. The induction of cytolytic T lymphocytes (CTLs) depends on recognition of foreign class II major histocompatibility complex antigens (human HLA-DR, DC/DS, SB and others, collectively referred to as Ia) on the target cells whereas killing by CTLs usually depends on recognition of foreign class I antigens (HLA-A, B), although some alloreactive CTLs recognize foreign Ia instead of HLA-A, B (refs 5-8). The expression of Ia antigens has traditionally been regarded as restricted to immunological cell types, and the presence of class II antigen-bearing 'passenger' leukocytes in rodent organ grafts appears necessary for graft rejection. Recently, Ia antigens have been observed by immunofluorescence microscopy on human renal and dermal capillary endothelium. We have previously shown that human umbilical vein endothelial (HUVE) cells in standard culture conditions do not bear Ia antigens, but may be induced to do so by products of lectin- or alloantigen-activated T lymphocytes. Furthermore, we found that recombinant immune interferon (IFN-gamma), free of other lymphokines, is a potent inducer of Ia expression in HUVE cells. Here we report that IFN-gamma also induces Ia expression on human foreskin capillary endothelial (HFCE) cells, HUVE cells transformed by Simian virus 40 viral DNA (SV-HUVE cells) and human dermal fibroblast (HDF) cells in culture. Further, we present evidence that Ia present on HUVE cells and HDF cells can be functionally recognized by human T cells, resulting in a two-way interaction between T cells and mesenchymal cells that may be important in allograft rejection.

Cells, Cultured↗

PubMedDate not supplied

Recombinant interleukin-2 directly augments the cytotoxicity of human monocytes.

Interleukin-2 (IL-2), originally described as a growth factor required for sustained proliferation of T cells in vitro is a glycoprotein hormone of known structure which appears to be important for the generation of immune responses in vivo. As well as T lymphocytes, B lymphocytes and large granular lymphocytes with natural killer activity (NK cells) can also respond to IL-2. The action of IL-2 seemed to be limited specifically to lymphocytes, however, and the term 'T-lymphocytotrophic hormone' was used. Here we provide evidence that human monocytes display a substantially increased cytotoxic activity as a direct and rapid response to human recombinant IL-2 but not to human recombinant glycosylated interferon-gamma (IFN-gamma) or lipopolysaccharide. Our results reveal a previously unknown function of IL-2 and suggest its possible involvement in monocyte-T cell interactions.

Cell Division↗

PubMedDate not supplied

Isolation and characterization of a human fibroblast interferon gene and its expression in Escherichia coli.

Human fibroblasts were induced for interferon synthesis, and the mRNA coding for interferon was partially purified. On this basis, double-stranded DNA copies were synthesized enzymatically and were inserted into cloning vehicles. A large collection of colonies containing chimeric plasmids was obtained. An RNA selection method was used for the identification of several individual clones containing the human fibroblast interferon (IFN-beta) cDNA. From the nucleotide sequence of the cloned structural gene, the primary structure of the mRNA and, hence, of the protein itself was deduced. IFN-beta is a polypeptide 166 amino acids long and contains a single site for N-glycosylation; IFM-beta is normally made as a preinterferon containing a signal sequence that is 21 amino acids long. The interferon gene was inserted into an expression plasmid under thermoinducible control of the phage lambda PL promoter. this allowed the synthesis of polypeptides in the bacteria, which for all physicochemical, biological, and immunologic properties tested closely resembled authentic IFN-beta.

Amino Acid Sequence↗