Certain infections and malignancies in mammals cause the development of a condition known as cachexia in which the animal continues to lose weight, often while consuming an adequate diet. When macrophages are stimulated with an endotoxin, they produce a factor or factors, termed cachectin, that inhibits the activity of fat-producing (lipogenic) enzymes in cultured adipocytes. This effect may reflect one of the physiological bases for cachexia. In the present study, clones of complementary DNA from genes whose expression is increased during the differentiation of adipocytes were used to study the molecular basis of cachectin's actions. In the presence of cachectin, the expression of the corresponding genes was reversibly and specifically inhibited. Furthermore, when mature adipocytes were exposed to cachectin, the messenger RNA's of those genes diminished and rapidly approached the levels present before differentiation.

Using Chinese hamster ovary (CHO) cells transfected with a plasmid carrying the human fl-interferon gene, we find that inhibitors of protein synthesis, in the absence of any other inducer, stimulate the production of interferon RNA; this effect is maintained in cells in which the plasmid sequences have been amplified 25-to 50-fold. Nuclear transcription assays show that a major effect of cycloheximide is to increase the rate of transcription of the interferon gene. This contradicts the generally accepted explanation that inhibitors of protein synthesis augment interferon production by stabilizing interferon mRNA. In addition, we have studied the effects of double stranded RNA [poly(rI)-poly(rC)] on the induction of interferon RNA in the presence and absence of cycloheximide. Our results indicate that poly(rI)-poly(rC) by itself causes a transient increase in interferon RNA; however, in the presence of cycloheximide this effect is prolonged. We do not, however, find an increase in transcription of the interferon gene(s) as an early response to poly(rI)-poly(rC). Finally, we hate found that cells treated with cycloheximide or infected with Newcastle disease virus induce large amounts of a secreted l1-kDa protein. This cellular protein is not inducible by poly(rI) poly(rC). We propose that both interferon and this 11-kDa protein belong to a family of proteins in which production is regulated in a coordinate fashion during viral inhibition of cellular protein synthesis.Interferons (IFNs) uCi/ml (1164 Ci/mmol; 1 Ci = 37 GBq; New England Nuclear) in serum and methionine-free Dulbecco's modified Eagle's medium. A 50-,u1 aliquot of the tissue culture supernatant was concentrated by drying under vacuum and then it was suspended in NaDodSO4/polyacrylamide gel sample buffer. After boiling for 5 min, the samples were electrophoresed on a 15% polyacrylamide gel according to the procedure of Laemmli (13). The gel was dried and exposed to xray film for 12 hr.

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Plasmid DNA containing the human beta-interferon (IFN-,B) gene and mouse dihydrofolate reductase cDNA was transfected into dihydrofolate reductase-negative Chinese hamster ovary cells. Dihydrofolate reductase-positive transformants were obtained, and cells containing amplified copies of mouse dihydrofolate reductase were selected by exposure to increasing methotrexate concentrations. These cells were found to express high levels of human IFN-4 after polyriboinosinic acid-polyribocytidylic acid superinduction or NDV infection; this was a result of coamplification of the IFN-P gene. Levels of expression of 1 U/cell per day were achieved on superinduction, giving corresponding titers of up to 1010 U/liter medium in culture supernatants. Constitutive production of IFN-P at rates of about 0.5% of superinduced rates was observed; cells producing these levels of IFN-1 had acquired resistance to cytotoxic antiviral effects of IFN-P. Two forms of human IFN-,B were produced; a major glycosylated 23,000-dalton form and an unglycosylated 18,500-dalton form. The latter had greatly reduced antiviral activity. IFN-P production was very sensitive to cellular growth rate; the highest levels were produced by density-arrested cultures. Regulation of IFN-,B production by polyriboinosinic acid-polyribocytidylic acid or by cell density effects required the presence of DNA sequences 5' to the IFN-,-coding sequences; replacement of these sequences with the simian virus 40 early promoter resulted in uninducible, density-independent production of IFN-P.Interferons are secreted polypeptides that protect cells from virus infection. Two classes of human interferon (IFN) have been described: type I IFNs (IFN-ot, IFN-P) are induced by viral infections and are acid stable and type II IFN (IFN--y) is induced after stimulation of T-lymphocytes and is acid sensitive. IFNs of both types have been purified to homogeneity, and their biochemical and biological properties have been studied extensively (19,26). Genes coding for these polypeptides have been cloned, and their nucleotide sequences have been determined (5,8,9,12,15,17,27 Fig. 1.Next, the 838-bp TaqI fragment of the IFN-P gene, which on May 12, 2018 by guest

We have used a mouse mammary tumor virus (MMTV)-infected rat hepatoma cell line as a model system for studying glucocorticoid action. These cells induce tyrosine aminotransferase and MMTV in response to the synthetic glucocorticoid, dexamethasone. The major viral antigen, a glycoprotein of 52,000 daltons (gp52), appears on the surface of infected cells in amounts which reflect the cytoplasmic content of viral RNA. Using an anti-gp52 antiserum and a fluorescence-activated cell sorter (FACS), we have selected variants which display low levels of pg52 in the presence of the hormone. Multiple cycles of enrichment for cells that fluoresce weakly in the presence of hormone have generated a population which fails to produce a detectable increase in cell surface gp52 in response to dexamethasone. This population of nonresponders and a number of independent clones derived from this population were analyzed for their ability to induce gp52 and TAT and for these presence of glucocorticoid receptors. All nonresponder clones exhibited little or no induction of either glucocorticoid-inducible marker. Two of the clones contained reduced levels of glucocrticoid receptor, while the remainder of the clones showed no detectable specific hormone binding. These results provide genetic evidence that a single class of glucocorticoid receptors is involved in the induction of both MMTV and TAT in HTC cells.