Biomedical subjects
Publications and source records attributed to S Pestka.
A murine alpha interferon gene (MuIFN-alpha A) was isolated from a murine genomic DNA library in phage lambda. The gene was cloned and its sequence determined. It was expressed in Escherichia coli under the control of the trp promoter/operator which resulted in antiviral activity on mouse L-cells. MuIFN-alpha A exhibits 60% homology to HuIFN-alpha A at the amino acid level.
The structural and functional complexity of the human interferon system has become increasingly evident. More than eight different alpha (leukocyte) interferons are expressed in induced human cells in culture. Many of these have been purified by a combination of methods, including high-performance liquid chromatography. Moreover, at least 12 different human leukocyte interferons have been cloned, and several have been efficiently expressed in Escherichia coli and other organisms. The availability of purified species of leukocyte interferon, both natural and recombinant, has allowed structural work to be done, including amino acid sequence determinations, chemical modification studies, and the crystallization of one species. The purified material has also been used for the production of monoclonal antibodies with various specificities that are proving invaluable in rapid assays and purification techniques. Testing of the purified species for their relative potency in antiviral, antiproliferative, and immunomodulatory assays has begun to demonstrate the functional uniqueness and diversity of the purified alpha interferons. Hybrid interferon genes have been synthesized by splicing together parts of various cloned interferon genes. The resulting hybrid proteins have been valuable in establishing structure/function relationships. In several cases, the functional properties of the hybrid protein were novel and unpredicted from the properties of the parental molecules.
The antiviral activities of recombinant human leukocyte interferons IFN-alpha A and IFN-alpha D as well as five hybrids of these interferons against retroviruses, vesicular stomatitis virus, and encephalomyocarditis virus were studied in feline, human, and murine cells. Although these interferon species had widely different potencies, their activities against these viruses were, in general, proportional. The IFN-alpha A/D (Bgl) hybrid was the most potent species, and the IFN-alpha D/A (Bgl) hybrid was the least potent. However, the latter species did not interfere with the action of the former species. Like natural human leukocyte interferon, each of the seven species of recombinant interferons induced the synthesis of at least five proteins in human fibroblasts, whereas induction of only one such protein was readily detected in a feline fibroblast line in which these interferon species inhibited the replication of all three viruses.
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Treatment of human breast or colon carcinoma cells with recombinantly derived human leukocyte (clone A) interferon (IFN-alpha A) increases the surface expression of specific tumor-associated antigens (TAAs) recognized by monoclonal antibodies (MAbs). The MAbs used, B1.1, B6.2, and B72.3, recognize three distinct TAAs, i.e., the Mr 180,000 carcinoembryonic antigen, a Mr 90,000, and a Mr 220,000 to 400,000 glycoprotein, respectively. The binding of the MAbs to the surface of tumor cells increased in a dose-dependent manner, with optimal levels of TAA enhancement at 100 to 1,000 units IFN-alpha A/ml. Higher concentrations of IFN-alpha A that were cytostatic or cytotoxic were also less effective in enhancing TAA expression. Human melanoma (A375) cells and normal fibroblasts (WI-38 and Flow 4000) do not express any of the three TAAs, either before or after interferon treatment. The ability of IFN-alpha A to increase the expression of TAAs on human carcinoma cells was also temporally dependent, with optimal enhancement occurring after 16 to 24 hr. The enhancement of specific TAAs at the surface of the carcinoma cells by IFN-alpha A was confirmed, using fluorescence-activated cell sorter analysis. These data demonstrate that the IFN-alpha A-mediated increase of surface antigen is a result of both an accumulation of more antigen per cell, and an increase in the percentage of cells expressing the antigen. The ability of recombinant interferon to enhance specific TAAs on human carcinoma cells may be exploited in designing protocols for the in situ detection and therapy of human carcinoma lesions by MAbs, as well as in further defining the role of specific TAAs in the expression of the transformed phenotype.
With a panel of monoclonal antibodies, the effect of recombinant human leukocyte interferons (i.e., IFN-alpha A, IFN-alpha D), of a hybrid leukocyte IFN (i.e., IFN-alpha A/D (Bg1)), and of recombinant fibroblast (beta) IFN on the expression and shedding of four types of melanoma-associated antigens (MAA) and of HLA antigens by the cultured melanoma cell line Colo 38 was investigated. None of the IFN affected the expression of the high m.w. melanoma-associated antigen (HMW-MAA), but all of them increased its shedding. The expression and shedding of the 115,000 MAA and of the 100,000 MAA were increased by IFN; the magnitude of the effect as well as the kinetics were different for the various IFN preparations. The cytoplasmic MAA was the most sensitive to modulation by IFN, because all four types increased its surface expression, its total content, and its shedding. The three types of leukocyte IFN, as well as the fibroblast IFN, were all effective in increasing the expression of HLA-A,B,C antigens, the effect being more marked on the free heavy chain than on the HLA-A,B,C complex. However, only leukocyte IFN enhanced the shedding of the HLA-A,B,C molecular complex. The three types of leukocyte IFN and especially fibroblast IFN enhanced the expression of the gene products of the HLA-D region, the effect being more marked on DC-1 antigens than on HLA-DR antigens. No effect on the shedding of HLA-DR and DC-1 antigens was detected.
Two recombinant human leukocyte interferons (A and D), five hybrid interferons containing varying portions of A and D, and one fibroblast recombinant interferon were tested over a wide range of concentrations for their ability to modulate cytolytic activity of human natural killer (NK) cells. All of the interferons tested were purified to homogeneity. Although all the interferons were active, there were significant quantitative differences in their ability to augment cytolysis and the rank order of potency was reproducible among donors. The various recombinant interferons were also tested for their ability to augment mouse NK activity and the parental D and the A/D hybrids exhibited significant augmentation of cytolysis, which was consistent with their interspecies reactivity in viral neutralization assays. There generally was a direct correlation between antiviral activity and the ability of interferon to augment mouse NK activity; however, this correlation was not evident when tested on human cells. The study of these hybrids led to the identification of two molecules (A/D Bgl and A/D Pvu) which are very active in augmenting mouse NK activity. In addition, considerable insight has been obtained regarding the structure-function relationship of these leukocyte interferons and their ability to boost murine NK. This biological activity was associated with the COOH-terminal portion of the D interferon.
There is good agreement between the target molecular weight and the known molecular weight of human leukocyte interferons (about 20,000). The target molecular weight of fibroblast interferon, 31,000 to 42,000, is significantly larger than the monomer molecular weight of 21,000 to 24,000, suggesting that the dimer may be the predominant active functional unit in solution. A range from 63,000 to 73,000 for the target molecular weight of several different fractions of immune interferon (including natural crude as well as the recombinant form) indicates that the functional form of the immune interferon may be a trimer or tetramer. Thus, these studies indicate that the functional unit of leukocyte interferon is the monomer, that of fibroblast interferon is a dimer, and that of immune interferon is probably a tetramer (or trimer).
A complementary DNA library prepared from the 12S polyadenylated RNAs extracted from interferon-induced KG-1 cells, a human myeloblast cell line, was screened for the presence of induction-specific sequences. Clones that exhibited strong positive signals were separated by hybridization criteria into nine classes. Clones from classes I through IV consisted of about 78% of the total and unexpectedly were found to resemble human mitochondrial ribosomal RNA genes.
An immunoassay is described that discriminates between monomers and oligomers of human leukocyte interferon. The assay in principle can be used to distinguish between monomers and oligomers of any substance.
Antiviral activity of recombinant human leukocyte A interferon was inactivated by heating at 65 degrees C or by reduction of disulfide bonds. The specific immunoreactivity, as measured by radioimmunoassays measuring binding to monoclonal antibodies, decreased concomitantly with the antiviral activity. Although the monoclonal antibodies did bind to inactivated interferon, their binding affinity to inactivated interferon was in general very much lower than their binding affinity to active interferon. Therefore, this immunoassay could replace the antiviral assay for detection of biologically active interferon. In addition, most of these antibodies should be especially useful for purification of the interferons since they discriminate between the native active and inactive denatured species. Screening for such antibodies is convenient and simple. The general use of antibodies that preferentially interact with native molecules provides a powerful new principle for choosing monoclonal antibodies with extraordinary potential in assay and purification.
Ten species of purified human leukocyte interferon were tested for their ability to modulate the cytolytic activity of natural killer (NK) cells and the cytolytic and cytostatic activities of monocytes. The interferon species were tested at several antiviral titers and examined for quantitative differences in their ability to modulate immunological function. At the higher doses of interferon (i.e., greater than 500 units) all of the interferon species demonstrated significant augmentation of cytolysis and cytostasis. However, when low levels (i.e., 10-50 units) of interferon were employed, appreciable differences between the various interferon species were seen. A similar pattern of relative potency among the various species of pure leukocyte interferon was seen for augmentation of cytolysis by monocytes and NK cells. In contrast, a different pattern of relative potency was observed for augmentation of cytostasis. These results demonstrated substantial quantitative differences (as much as 100-fold) in the ability of the various species of human leukocyte interferon to induce significant levels of augmentation of these cell-mediated functions. Such results should have significant impact in choosing a specific interferon species for appropriate clinical trials.
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A recombinant plasmid was constructed that results in deletion of eleven COOH-terminal amino acids of human leukocyte A interferon and their replacement by nine unrelated amino acids encoded by the beta-lactamase gene of pBR322. This altered human leukocyte interferon exhibits antiviral activity slightly lower than the natural molecule and appears to be more susceptible to proteolytic degradation as well. These results also confirm the conclusion that the eleven COOH-terminal amino acids are not essential for antiviral or antiproliferative activity.
We have studied the effects of human leukocyte interferon produced in bacteria and diterpene phorbol ester tumor promoters on differentiation of normal human myoblast cultures derived from mature skeletal muscle. Interferon (100-5,000 units/ml) induced an acceleration of myotube formation and creatine kinase (CK; EC 2.7.3.2) isoenzyme transition from CK-BB to CK-MM. Heat-inactivated or trypsin-treated interferon did not affect the differentiation process. In contrast, the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), but not its inactive structural analogues phorbol and 4 alpha-phorbol 12,13-didecanoate, caused a dose-dependent (0.01-100 ng/ml) inhibition of myotube formation and CK isoenzyme transition. Neither interferon nor TPA had a significant effect on myoblast proliferation prior to fusion, and the cloning efficiencies were similar as well. Opposing effects of interferon and TPA were also demonstrated by simultaneous application of these agents to the cultures. These studies suggest that some of the antitumor effects of interferon may relate to its capacity to modulate cellular differentiation.
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