Marie-Thérèse Bandu

Identification of the cell multiplication inhibitory factors in interferon preparations as interferons

THERE has been some controversy for the past few years concerning the inhibitory effect of interferon on cell multiplication. We and others have presented evidence that interferon itself is the responsible factor1–13. Some investigators have, however, failed to observe any inhibition of cell multiplication with interferon preparations14–17; while others have observed inhibition but ascribed this effect to substances other than interferon in their preparations, either because no inhibition was observed when more purified preparations were used18 or because of apparently inconstant ratios between antiviral and cell multiplication inhibitory (CMI) activities in a given preparation19–21. It has been claimed that the factor responsible for the CMI activity of human leukocyte interferon preparations could be physically separated from the factor responsible for the antiviral activity22. We provide strong evidence that the antiviral and CMI activities of interferon preparations are indeed intrinsic properties of the covalent structure of interferons.

Specific high-affinity binding of 125I-labeled mouse interferon to interferon resistant embryonal carcinoma cells in vitro

Abstract Multipotential mouse embryonal carcinoma cells are resistant to several biologic effects of mouse interferon: inhibition of viral multiplication and inhibition of cell division. Nevertheless using 125 I-labeled highly purified mouse interferon we have shown that these embryonal carcinoma cells express specific interferon receptors in similar number and of the same affinity as interferon sensitive differentiated embryonal carcinoma cells. Thus, mechanisms of interferon resistance are probably multiple: some cells are resistant because they lack specific binding sites for interferon (interferon resistant mouse L1210 cells) and other cells, as shown herein, are resistant to some of the effects of interferon despite binding of interferon to specific receptor sites. Furthermore, these binding sites may be considered functional, since interferon does induce an increase in 2-5A synthetase in these cells.

Specific Binding of High-Mobility-Group I (HMGI) Protein and Histone H1 to the Upstream AT-Rich Region of the Murine Beta Interferon Promoter: HMGI Protein Acts as a Potential Antirepressor of the Promoter

ABSTRACT The high-mobility-group I (HMGI) protein is a nonhistone component of active chromatin. In this work, we demonstrate that HMGI protein specifically binds to the AT-rich region of the murine beta interferon (IFN-β) promoter localized upstream of the murine virus-responsive element (VRE). Contrary to what has been described for the human promoter, HMGI protein did not specifically bind to the VRE of the murine IFN-β promoter. Stably transfected promoters carrying mutations on this HMGI binding site displayed delayed virus-induced kinetics of transcription. When integrated into chromatin, the mutated promoter remained repressed and never reached normal transcriptional activity. Such a phenomenon was not observed with transiently transfected promoters upon which chromatin was only partially reconstituted. Using UV footprinting, we show that the upstream AT-rich sequences of the murine IFN-β promoter constitute a preferential binding region for histone H1. Transfection with a plasmid carrying scaffold attachment regions as well as incubation with distamycin led to the derepression of the IFN-β promoter stably integrated into chromatin. In vitro, HMGI protein was able to displace histone H1 from the upstream AT-rich region of the wild-type promoter but not from the promoter carrying mutations on the upstream high-affinity HMGI binding site. Our results suggest that the binding of histone H1 to the upstream AT-rich region of the promoter might be partly responsible for the constitutive repression of the promoter. The displacement by HMGI protein of histone H1 could help to convert the IFN-β promoter from a repressed to an active state.

Synergism between Multiple Virus-induced Factor-binding Elements Involved in the Differential Expression of Interferon A Genes

Comparative transfection analysis of murine interferon A4 and interferon A11 promoter constructs transiently transfected in mouse L929 and human HeLa S3 cells infected with Newcastle disease virus showed that the second positive regulatory domain I-like domain (D motif), located between nucleotides −57 and −46 upstream of the transcription start site, contributes to the activation of virus-induced transcription of the interferon (IFN)-A4 gene promoter by cooperating with the positive regulatory domain I-like and TG-like domains previously described. Electrophoretic mobility shift assay performed with the virus-inducible fragments containing these motifs indicated that the binding activity that we have denoted as virus-induced factor (Génin, P., Bragança, J., Darracq, N., Doly, J., and Civas, A. (1995) Nucleic Acids Res. 23, 5055–5063) is different from interferon-stimulated gene factor 3. It binds to the D motif but not to the virus-unresponsive form of the D motif disrupted by a G−57 → C substitution. We show that the low levels of IFN-A11 gene expression are caused essentially by the lack of two inducible enhancer domains disrupted by the A−78 → G and the G−57 → C substitutions. These data suggest a model taking account of the differential regulation of IFN-A gene family members. They also suggest that virus-induced factor may correspond to the primary transcription factor directly activated by virus that is involved in the initiation of IFN-A gene transcription.

Interferon and Cell Division VII. Inhibitory Effect of Highly Purified Interferon Preparations on the Multiplication of Leukemia L 1210 Cells

Summary Highly purified mouse interferon preparations prepared by different techniques in different laboratories inhibited the mul–tiplication of mouse leukemia L 1210 cells. The dose–response curves of the antiviral activity of interferon and the anticell multiplication activity using a sensitive assay were parallel. Likewise, the kinetics of the appearance of the factor responsible for both activities and the curves of thermal inactiva–tion of this factor were also parallel. It seems reasonable to conclude that interferon itself can inhibit cell multiplication. I. G. is deeply indebted to Dr. Sidney Farber, Director of the Childrens Cancer Research Foundation, Boston, MA for his continued interest and support. K. P. acknowledges the aid of NSF Grant No. G. B.–26515. M. T. is the recipient of an EMBO fellowship.

Indomethacin and aspirin do not inhibit the antiviral or anti-proliferative actions of interferon.

Neither indomethacin nor aspirin, at concentrations which inhibited the formation of prostaglandins and prevented the interferon-induced increase in the intracellular concentration of cyclic GMP, had any significant effect on the development of the interferon-induced antiviral state either in mouse L1210 cells challenged with vesicular stomatitis virus or in mice infected with encephalomyocarditis virus. Furthermore, neither drug had any significant effect on the interferon-induced inhibition of cell multiplication in cultures of mouse leukaemia L1210 cells. The differences in the effects of these cyclo-oxygenase inhibitors on different interferon effects may provide some insight into the different pathways of interferon action.

Murine interferon-alpha1 gene-transduced ESb tumor cells are rejected by host-mediated mechanisms despite resistance of the parental tumor to interferon-alpha/beta therapy.

The highly metastatic ESb tumor is totally resistant to murine interferon-α/β (IFN-α/β) therapy, regardless of the number of cells injected or the route of inoculation. In contrast, as we show herein, mouse IFN-α1-transduced ESb tumor cells were inhibited markedly when injected subcutaneously into immunocompetent mice. IFN-producing ESb tumor rejection was mediated by the immune system, because these tumor cells grew normally in immunosuppressed mice. Tumor regression was accompanied by extensive necrosis and cellular infiltrates in the tumor area. These results further support the use of IFN-α in cytokine gene therapy of cancer and suggest the advantage of using gene transfer rather than cytokine administration to enhance an antitumor immune response.