Chick interferon: heterogeneity of electric charge.

Chick interferon from allantoic fluid, virus-induced and partly purified, consists of several active components of different charge. The components are separable by elution at different pH values from a carboxymethyl-Sephadex column; they also occupy different pH zones in electrofocusing gradients. Most of the interferon is, however, isoelectric near neutrality.

Positive interactions between interferon and chemotherapy due to direct tumor action rather than effects on host drug-metabolizing enzymes.

The mechanism of increased antitumor activity when human lymphoblastoid interferon [HuIFN-alpha(Ly)] and the drugs cyclophosphamide and Adriamycin are used in combination on a human tumor xenograft in nude mice has been investigated. HuIFN-alpha(Ly) did not affect hepatic levels of the drug-metabolizing enzymes cytochrome P-450 or the glutathione S-transferases. In contrast, mouse interferon caused significant and differential changes in the isozymic forms of these enzymes. However, addition of mouse interferon to the HuIFN-alpha(Ly)/cyclophosphamide or Adriamycin combinations had no effect on the final result, and did not increase the toxicity of the combination therapy. These data provide evidence that the increased activity of the combination therapy is due to effects on the tumor rather than on the host. Further studies showed significant perturbations in the tumor cell cycle after in vivo combination therapy. Cyclophosphamide caused an accumulation in G2 and the addition of HuIFN-alpha(Ly), which alone caused little change in cycle distribution, delayed this G2 block and strongly increased the number of cells in S phase. A similar, although less pronounced, effect was seen with HuIFN-alpha(Ly)/Adriamycin therapy. The increase in S phase seen in combined therapy may account for the synergy seen.

Interferons: purification and physicochemical aspects.

Antiviral interferon activity in any one species can be exhibited by a variety of substances that differ in their physical and chemical properties, but the nature of these differences is not understood. Conditions that can lead to the formation of diverse types of interferons have been outlined. Reasons have been adduced why, for certain purposes, purification of interferons is desirable or even necessary, and examples have been presented to show how and to what extent this has been achieved. In spite of some very high purification factors, not a single interferon has been obtained as a pure substance. Therefore, all available knowledge of physical and chemical properties has been obtained by indirect means.

Chemical modifications of human and murine interferons.

Methods that modify the carbohydrate and peptide portions of human and murine interferons have been described. Such work has helped considerably in our understanding of these fascinating substances. The most powerful of the methods relies on gene manipulation: it has already led, and will lead in the future, to further species with altered biological activities. Some of them may prove clinically advantageous, but one will have to be aware of the fact that some of these "unnatural" interferons could lead to immunological complications.

Interferons: chemical properties.

HuIFN-alpha has been found to consist of a family of 8-10 components. Their amino acid (165 and 166 residues) and nucleotide sequences are similar and also reveal some relationship to that of HuIFN-beta and even to those of murine alpha- and beta-interferons. It is thus likely that all mammalian interferons have originated from a common ancestral gene. Human beta- and some alpha-interferons have been obtained by genetic engineering in E.coli. All interferons seem to contain a relatively high proportion of hydrophobic amino acids and some of the components are glycosylated. The N-terminal residue of all HuIFN-alpha members is probably cysteine, that of the major HuIFN-beta peptide methionine and that of MuIFN-alpha and-beta probably alanine and isoleucine. Very little is known about the chemistry of any of the gamma-interferon species.

Specific activity of pure human interferons and a non-biological method for estimating the purity of highly purified interferon preparations.

Methods for determining the specific activity and percentage purity of highly purified interferon preparations are unreliable, mainly because of the difficulty in estimating very small quantities of protein. Human leukocyte-derived interferon (HuIFN-alpha) is a mixture of several distinct species, and the relationship between specific activity and purity is not direct. A non-biological method is described here, by which the purity of highly purified HuIFN-alpha is determined chromatographically. Samples are run on a Sephadex G-75 column with continuous measurement of E206 in the eluate, and SDS gel electrophoresis is used to separate essentially all the protein species in the characteristic double peak region containing interferon activity and show that they are interferons. The validity of the method has been checked using a range of standard proteins. The specific activity of pure Namalwa (lymphoblastoid) HuIFN-alpha was estimated to be 1.0--1.7 x 10(8) U/mg protein.

A family of structural genes for human lymphoblastoid (leukocyte-type) interferon.

Amino acid sequences of tryptic and chymotryptic peptides from human lymphoblastoid interferon (IFN-alpha) have been determined. The results show that IFN-alpha consists of a family of proteins with at least five different, but homologous, primary structures. There appears to be little, if any, glycosylation of the major components of IFN-alpha.

Purification of poliovirus by affinity chromatography.

Poliovirus type 1 (Mahoney strain) has been purified by retention on a Sepharose 4B-antibody column and elution with 3M K thiocyanate. The virus was recovered in excellent yield and its purity was as high as that achieved by detergent treatment followed by sucrose gradient centrifugation. The column could be re-used and its capacity was sufficiently high to make it a useful method for the purification of milligram quantities of the virus.

Human lymphoblastoid cells as a source of interferon.

Interferons have considerable antitumour effects in animals, and have been used with encouraging results in patients with osteocarcomas and certain other tumours. So far only relatively small amounts of material suitable for use in man have been prepared, and almost all of this has come from human white blood cells [buffy coats]. Human fibroblast cell lines are now increasingly being used as an alternative source, but the resultant interferon differs in its chemical and biological properties from leucocyte interferon. Lymphoblastoid cells can also be induced to form an interferon which appears identical to buffy coat interferon. These cells can be grown in suspension in large tanks, and could provide large amounts of relatively inexpensive interferon. The advantages of this type of production system and the problems associated with it will be discussed.

Human interferon inhibits the growth of established human breast tumours in the nude mouse.

In this paper we describe a model system for looking at the effects of human interferon, IFN, on an established human tumour. Highly purified human IFN derived from lymphoblastoid cells (HuIFN alpha-Namalwa) strongly inhibited the growth of a human breast cancer growing as a xenograft in nude mice. The effect was dose-dependent, required daily treatment for an optimal effect and was time-dependent, little inhibition being seen before 2 weeks of therapy. With the doses used, however, neither tumour regression nor disappearance were seen and on morphological examination, treated tumours appeared as miniatures of control tumours. The inhibition of the tumour by HuIFN alpha-Namalwa appeared to be due to a direct effect on the human cells as this IFN had little effect on the mouse immune system in vitro as measured by NK cells activity. Also HuIFN therapy had no effect on levels of an interferon induced enzyme, 2-5A synthetase, in the mouse spleen cells but stimulated this enzyme in the human tumour.

Ribonuclease activity of preparations of human lymphoblastoid interferon.

Crude human lymphoblastoid interferon has less ribonuclease activity than equivalent primary leukocyte interferon and ribonuclease was eliminated when it was purified. The methods used differed from those that had failed to eliminate similar activity from leukocyte interferon. This result makes it unlikely that exogenous ribonuclease plays a major role in the antiviral action of interferon preparations.

Interferon action on parental Semliki forest virus ribonucleic acid.

Actinomycin D-treated chick fibroblasts were infected with purified (32)P-labeled Semliki forest virus, and ribonucleic acid (RNA) was extracted after 1 or 2 hr. Within 1 hr, viral RNA forms sedimenting in sucrose gradients at 42S, 30S, and 16S were present. The 42S form corresponded to the RNA of the virion. The 16S form appeared to be a double-stranded template for the formation of new viral RNA, since nascent RNA was associated with it and the molecule could be heat-denatured and subsequently reannealed by slow cooling. Interferon treatment before infection, or puromycin (50 mug/ml) or cycloheximide (200 mug/ml) added at the time of virus infection, had no effect on the formation of the 30S RNA but inhibited the production of the 16S form. Several findings made it unlikely that these results were due to breakdown of parental RNA and reincorporation of (32)P into progeny structures. The results suggested that the mechanism of interferon action involves inhibition of protein synthesis by parental viral RNA, since a specific viral RNA polymerase had previously been demonstrated to be necessary for production of 16S RNA. No protein synthesis appears necessary for formation of 30S RNA from parental virus RNA.

Effects of HuIFN-alpha 2 and HuIFN-alpha (Namalwa) on breast cancer cells grown in culture and as xenografts in the nude mouse.

The activity of HuIFN-alpha 2 produced in bacteria has been compared with that of HuIFN-alpha from Namalwa cells (HuIFN-alpha N) as an antiviral (against EMC virus) and antiproliferative agent in normal and malignant cultured human breast cells. The IFN preparations show the same spectrum of antiviral and antiproliferative activity in the human cell strains and lines examined, and are equally effective, when comparable amounts of IFN protein are used. Both interferon preparations inhibit virus growth (EMC) in four types of bovine cells but neither inhibit the growth of these cells. HuIFN-alpha 2 and HuIFN-alpha N can also be shown to effectively inhibit the growth of a transplantable human breast cancer, grown as xenografts in the nude mouse, although the amounts of IFN protein required may not be the same for both preparations.

Analysis and purification of human lymphoblastoid (Namalwa) interferon using a monoclonal antibody.

Highly purified interferon-alpha (IFN-alpha) prepared from a human lymphoblastoid line (Namalwa) was analysed by gel filtration and polyacrylamide gel electrophoresis (PAGE). Gel filtration separated the IFN-alpha into two peaks (A and B). All the components of peak A were retained by a monoclonal antibody (NK2) column, but some of those from peak B were not retained. The IFN that was not bound was active on mouse cells and could be resolved into two major bands by PAGE. The bound fraction (about 75% of the interferon protein) was purified by means of the monoclonal antibody column, although complete purification of crude interferon was not achieved in one passage.