U-373 MG glioblastoma and IMR-32 neuroblastoma cell lines express the dopamine and vesicular monoamine transporters.

The U-373 MG glioblastoma and the IMR-32 neuroblastoma cell lines were found to express the dopamine (DA) and vesicular monoamine transporters, using reverse transcriptase-polymerase chain reaction (RT-PCR). To further characterize the DA transporter, [3H]GBR-12935 binding and [3H]DA uptake studies were performed. Specific binding of [3H]GBR-12935 to U-373 MG and IMR-32 cells is saturable as saturation experiments indicated. Scatchard analysis revealed two binding sites on U-373 MG as well as on IMR-32 cells. The high-affinity sites exhibited a KD of 2.95 and 0.42 nM and a Bmax of 6.4 and 0.83 fmol/mg protein for U-373 MG and IMR-32 cells, respectively. The low-affinity sites exhibited a KD of 144 and 251 nM and a Bmax of 37.5 and 119 fmol/mg protein for the same cells, respectively. The high-affinity binding of both types of cells probably represents the "classic" DA uptake site identified in other studies from human and rat striatal membranes or synaptosomes, while the low-affinity binding may represent a mazindol-insensitive binding site (the "piperazine acceptor site"). [3H]DA uptake was 0.55 +/- 0.16 and 1.08 +/- 0.33 pmol/mg protein for U-373 MG and IMR-32 cells, respectively. Since the DA transporter has been implicated as an important site for drugs and toxins, the above-mentioned cell lines may be a useful tool in the study of the mechanism of action of DA transporter modulating substances.

Synergistic stimulatory effects of tumour necrosis factor alpha and interferon gamma on replication of human immunodeficiency virus type 1 and on apoptosis of HIV-1-infected host cells.

Differential and sometimes contradictory effects have been described for tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) on replication of human immunodeficiency virus type 1 (HIV-1). The authors examined individual and coordinate action of these cytokines on HIV-1 expression, and on apoptosis of HIV-1-infected host cells by determination of reverse transcriptase activity in cell culture supernatant, expression of HIV-1-RNA and production of p24 antigen in the promonocytic cell line U937 and its persistently HIV-1-infected clone U1. Apoptosis was demonstrated by typical cleavage of cellular DNA at internucleosomal regions in promonocytic and T-lymphocytic cell lines. TNF-alpha alone markedly stimulated HIV-1 replication in U1 cells at the transcriptional and on the translational level. Exclusive application of IFN-gamma only slightly enhanced HIV-1 expression, whereas it synergistically potentiated stimulatory effects of TNF-alpha. Both cytokines also synergistically induced apoptosis in HIV-1-infected host cells. Co-ordinate action of TNF-alpha and IFN-gamma is suggested to represent an important mechanism for disease progression in HIV infection. These findings demonstrate that cytokine effects on viral expression may vary depending on their single or combined application.

Chicken interferon consensus sequence-binding protein (ICSBP) and interferon regulatory factor (IRF) 1 genes reveal evolutionary conservation in the IRF gene family.

Members of the IRF family mediate transcriptional responses to interferons (IFNs) and to virus infection. So far, proteins of this family have been studied only among mammalian species. Here we report the isolation of cDNA clones encoding two members of this family from chicken, interferon consensus sequence-binding protein (ICSBP) and IRF-1. The predicted chicken ICSBP and IRF-1 proteins show high levels of sequence similarity to their corresponding human and mouse counterparts. Sequence identities in the putative DNA-binding domains of chicken and human ICSBP and IRF-1 were 97% and 89%, respectively, whereas the C-terminal regions showed identities of 64% and 51%; sequence relationships with mouse ICSBP and IRF-1 are very similar. Chicken ICSBP was found to be expressed in several embryonic tissues, and both chicken IRF-1 and ICSBP were strongly induced in chicken fibroblasts by IFN treatment, supporting the involvement of these factors in IFN-regulated gene expression. The presence of proteins homologous to mammalian IRF family members, together with earlier observations on the occurrence of functionally homologous IFN-responsive elements in chicken and mammalian genes, highlights the conservation of transcriptional mechanisms in the IFN system, a finding that contrasts with the extensive sequence and functional divergence of the IFNs.

Interferon sensitivity of expression of histone H5/H1(0)-vaccinia thymidine kinase fusion genes expressed by recombinant vaccinia viruses is enhanced by shortening the histone sequence.

To elucidate the structural basis responsible for the reduced IFN sensitivity of expression of the histone H1(0) and H5 gene, integrated into the vaccinia virus genome, vaccinia virus thymidine kinase (VV-TK)-histone H1(0)/H5 fusion genes were constructed and translocated into the TK locus of the VV genome. The chimeric genes, consisting of parts of either of the two histone genes and the 5' or 3' half of the TK gene, respectively, were expressed as histone-TK fusion proteins under the control of either the VV-TK promoter or the early sequences of the VV 7.5K promoter. IFN sensitivity of the expression of histone-TK fusion genes was shown to be influenced by the relative length of the histone sequence. Expression of fusion genes containing more than 45% cellular sequence either from the 5' or the 3' part of one of the two histone genes showed clearly reduced IFN sensitivity compared to the expression of VV-TK. On the other hand, by further reducing the relative amount of histone H5 or H1(0) sequence to 32%, the IFN sensitivity of expression of the corresponding fusion gene was drastically enhanced to levels indistinguishable from those of VV-TK.

Expression of authentic vaccinia virus-specific and inserted viral and cellular genes under control of an early vaccinia virus promoter is regulated post-transcriptionally in interferon-treated chick embryo fibroblasts.

The interferon sensitivity of the expression of an influenza-virus hemagglutinin (HA) gene cloned into the thymidine kinase (TK) gene of vaccinia virus was studied in chick embryo fibroblasts (CEF) and Madin-Darby bovine kidney (MDBK) cells. In CEF, the expression of the HA gene is inhibited by pretreatment of cells with homologous interferon. In MDBK cells, on the other hand, expression of the HA is not impaired by pretreatment with human interferon-alpha, and the synthesis of early vaccinia virus enzymes was also unaffected. These results indicate that the interferon sensitivity of HA gene expression is at least in part controlled by flanking regions of vaccinia virus DNA. In this report, we also address the question whether the expression of an influenza virus HA gene and the human histone H1 zero gene under control of a vaccinia virus immediate early promoter is affected in interferon-treated CEF by a post-transcriptional mechanism in the same way as the expression of the viral TK gene. In interferon-treated cells mRNA synthesis specific for all these genes was enhanced. Steady state mRNA levels 6 hr p.i. were, however, lower than the amounts expected from the rate of mRNA synthesis during the first 6 hr p.i., suggesting that part of the viral RNA was degraded. Degradation resistant mRNA accumulated in the interferon-treated cells in an amount comparable to that found in infected CEF. This RNA could be translated into viral protein in a cell-free system. Therefore the degradation of viral mRNA cannot solely be responsible for the inhibition of viral protein synthesis in interferon-treated cells.

Regulation of histone H5 and H1 zero gene expression under the control of vaccinia virus-specific sequences in interferon-treated chick embryo fibroblasts.

The duck histone H5 and human H1 zero were inserted into the thymidine kinase (TK) gene of vaccinia virus and the interferon sensitivity of their expression under the control of the viral TK and P7.5 promoters in chick embryo fibroblasts (CEF) was compared to the interferon sensitivity of vaccinia virus WR specific TK induction. Expression and transport of these histones to the nucleus in CEF infected with the appropriate vaccinia virus recombinants could be detected with antisera raised against chick histone H5. In CEF cultivated for 3 days, interferon treatment that completely inhibited TK synthesis had no or only a marginal inhibitory effect on the expression of the histone genes. Inhibition of the expression of the histones could be detected under conditions of increased interferon sensitivity in aged CEF. The magnitude of inhibition was, however, less pronounced than the inhibition of viral TK synthesis. These data indicate that flanking vaccinia virus DNA regions confer interferon sensitivity to the expression of these histone genes, but that they contain structural information that partially exempts their expression from the inhibitory activity of the interferon-induced regulatory system.

Two interferon sensitive steps in the replication cycle of Rous sarcoma virus.

De novo infection of Rous sarcoma virus (RSV) strains of receptor subgroups A, B, C, and D is inhibited by low doses of chick interferon. Adsorption and penetration into the cell are not marginally impaired by interferon treatment. Since the level of proviral DNA synthesis is strongly reduced in the interferon-treated cell it is concluded that uncoating or reverse transcription of the viral genome is inhibited. This inhibition of proviral DNA synthesis is not caused by an arrest of CEF in the stationary phase. Chronic infection of SR-RSV-A, -B, -C, but not SR-RSV-D is also sensitive to interferon. Chick interferon treatment (50 u/ml) also had no inhibitory effect on the amount of transcripts of the RSV-specific oncogene src or the cellular oncogenes src and myc.