Interferon-gamma priming effects in the activation and deactivation of ISGF3 in K562 cells.

ISGF3 is a major protein factor which mediates the transcriptional activation of interferon-inducible genes. ISGF3 is composed of two subunits, ISGF3 gamma and ISGF3 alpha, which are stimulated by interferon-gamma (IFN-gamma) and interferon-alpha (IFN-alpha), respectively. In this paper, the effect of pretreatment of IFN-gamma on the response of K562 cells to IFN-alpha, termed "gamma-priming," is examined. Using techniques of gene transfection and mobility shift assay, we studied the gamma-priming effects on the kinetics of appearance and disappearance of (i) the protein product of a luciferase reporter gene driven by an IFN-inducible promoter and (ii) the binding of ISGF3 to the interferon-stimulated response element. We found that exposure of cells to IFN-gamma prior to IFN-alpha greatly increased both the levels and the rate of ISGF3 binding activity during the early phase of IFN-alpha treatment and caused the amount of ISGF3 bound to the interferon-stimulated response element to decrease more rapidly with time during the later phase. Such effects were reflected also on the kinetics of expression of the interferon-inducible luciferase gene induced by IFN-alpha. In order to understand the basis of these gamma-priming effects, we use an in vitro reconstitution method to examine the kinetics of the subcomponents of ISGF3 in response to different IFN treatments in K562 cells. It was found that gamma-priming not only increased the levels of ISGF3 gamma, but it also stimulated both the rates of rise and fall of the activated alpha-component of ISGF3. A molecular model is proposed to explain these findings.

A novel interferon-inducible domain: structural and functional analysis of the human interferon regulatory factor 1 gene promoter.

We have cloned and functionally characterized the human interferon regulatory factor 1 (IRF-1) gene promoter. The promoter contains a CpG island, with several GC boxes, a CAAT box, but no TATA box. IRF-1 mRNA is strongly induced by gamma interferon (IFN-gamma) but more weakly and transiently by IFN-alpha. There are several putative kappa B motifs and numerous AA(G/A)G(G/T)A and GAAANN motifs throughout the promoter. The IRF-1 promoter is not autoregulated by the IRF-1 gene product. IFN inducibility of the promoter was studied with 5' deletion mutants linked to a heterologous reporter gene. Gel mobility shift assays were used to show IFN-inducible factor binding to the IRF-1 promoter. These studies showed that IFN inducibility is conferred by a novel imperfect inverted-repeat arrangement of two GAAANN motifs within a domain, 130 nucleotides upstream of transcription initiation. This inverted repeat binds a factor upon induction with IFN and can confer IFN inducibility on a heterologous promoter. Conversely, point mutations of the inverted repeat are not IFN inducible when linked to the same heterologous promoter.

High efficiency gene transfection by electroporation using a radio-frequency electric field.

In order to develop a safe and effective way to introduce exogenous genes into cells, we have experimented with a new method of electroporation which uses a radio-frequency (RF) electric field to permeabilize the cell membrane. This RF method has several advantages over the conventional electroporation method which uses a direct current (DC) field. We have shown that the RF electroporation method can be used to introduce marker genes into a wide variety of cell lines, including COS-M6, CV-1, CHO, 3T3 and hepatocytes, and is able to increase substantially the efficiency of gene transfection. (For example, the amount of DNA required for transfecting two million COS-M6 cells can be as low as 0.1 microgram). The transfection efficiency is shown to be affected by a number of factors, including cell type, field strength, pulse protocol and medium buffer. Because of its wide range of applications, high transfection efficiency and lack of harmful side-effect, the RF electroporation method would be particularly useful for introducing genes into human cells for gene therapy.

Effects of pH on cell fusion induced by electric fields.

Electrofusion has recently become an important area of cell biology research. We studied the effects of pH of the cell medium on the electrofusion of human red blood cells. Cell fusion was monitored by observing the movement of a lipophylic dye between neighboring fused cells using a fluorescence microscope. The cells were first brought into close contact by dielectrophoresis. Fusion was then induced by three pulses of high-intensity electric field. Within minutes following the pulse application, many cells were observed to fuse together to form fusion chains of different lengths. We found that the optimal pH for cell fusion is around pH 7.5. At this pH, the fusion yield was highest (ranging from 57 to 81%) and the average number of cells within a fusion chain was also the largest. The dependence of cell fusion on pH is more sensitive at low than at high pH. The fusion yield was decreased by 40% when the pH was changed from 7.5 to 6.0, but there was only a 20% decrease in yield between pH 7.5 and 10.0. We suspect that the observed pH effects may be caused by a redistribution of fixed charges at the cell surface, or changes in amphipathicity of the surface proteins.