Biomedical subjects
Publications and source records attributed to W A Carter.
Resting Burkitt's lymphoma cells (Daudi) in culture are more sensitive targets for the antiproliferative activity of purified human fibroblast interferon than cells that are rapidly multiplying. Thus, interferon may be of significant clinical value in neoplasms involving stem cells and, after chemotherapy, in suppressing the reemergence of tumors.
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A 23 year old woman with chronic active hepatitis documented by liver biopsy demonstrated persistent hepatitis B surface antigen, hepatitis B virus specific DNA polymerase hepatitis B core antigen (HBcAg), for approximately one year. The number of circulating T lymphocytes that rosetted with sheep erythrocytes was decreased, and a rosette-inhibitory factor was present in her peripheral blood. Interferon treatment (1 X 10(6) U/day intramuscularly for 82 days) resulted in a decrease of HBsAg and disappearance of HBcAg, (HBeAg) and specific DNA polymerase. In addition, the number of T lymphocytes increased to normal, and the rosette-inhibitory factor disappeared from the circulation. These findings suggest that the effect of interferon in chronic active hepatitis is mediated in part through its action on the immune system.
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Human interferon from normal diploid fibroblasts, purified by sequential chromatography on concanavalin A-agarose and phenyl-sepharose, was administered parenterally in 4 subjects. Fever, marked skin hypersensitivity reactions and suppression of marrow stem cells (estimated by the count of myeloid colony-forming cells), side-effects common for less purified fibroblast and leukocyte interferons, were absent. Purified fibroblast interferon retained antiviral and immunomodulatory activity, evidenced by reduction of the blastogenic response of peripheral lymphocytes and decrease of hepatitis B virus markers in a patient with chronic hepatitis B infection treated with this substance.
Some of the human prostatic cells in culture apparently produce oncornavirus-like particles. Bromodeoxyuridine does not enhance the production of these particles. On the contrary, this drug depresses such production. This depression is likely to be due to the cytotoxic effects of bromodeoxyuridine for these cells. These results can be interpreted to suggest that the human prostatic cells used in this study do not contain endogenous oncornavirus genetic information that is inducible. Purified human interferon inhibits the production of oncornavirus-like particles by these prostatic cells. It is also inhibits the rate of cellular DNA synthesis in these cells. These results are consistent with the notion that the inhibitory effects of interferon are mediated through its effect on cellular biosynthetic machinery.
Explant cultures from several human prostatic tissues have been examined for oncornavirus production. Some of these explants, epithelial in morphology, appear to release oncornavirus-like particles. The extracellular particulate material, obtained from the culture medium of these explants display the following characteristics: (1) It contains particles that band at a density of 1.1-1.2 g/cm3 in a sucrose density gradient. (2) The particles contain RNA-directed DNA polymerase. This polymerase utilizes poly(Cm) as a template and shows preference for poly(A) over poly(dA) and manganese over magnesium. (3) Apparently, the particles also contain RNA that directs the synthesis of DNA in vitro. The DNA thus synthesized is associated with RNA, some of it with high molecular weight RNA. Such particles are not observed in the culture medium from monolayers of monkey kidney CV-1 cells. We have found that explant cultures from 5 out of 15 tissue specimens with prostatic hyperplasia and 3 out of 4 with prostatic adenocarcinoma release particles that band at a density of 1.1-1.2 g/cm3 and contain RNA-directed DNA polymerase. Explant cultures of two normal prostates examined did not release such particles.
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Interferon, a glycoprotein with demonstrated antitumor and antiviral properties, can be obtained from cells of different species, and some of these interferons can be effective across species lines. I hypothesize that the ability to cross or not to cross species lines lies in the carbohydrate moiety, and cross-species biological activity is a property of the polypeptide. This would represent one of the first major roles uncovered for glycosylation in any biological system. If this hypothesis is correct, then it should be possible to use animal-derived interferon as a large scale, inexpensive source to treat human diseases.
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Human immune interferon, induced in leukocytes by phytohemagglutinin, was prepared in the absence and presence of tunicamycin, an antibiotic which selectively inhibits the glycosylation of newly synthesized glycoproteins. Interferon preparations, produced in the absence of the antibiotic, displayed a considerable chromatographic heterogeneity on: (a) concanavalin A-agarose, (b) phenyl-agarose, (c) Cibacron Blue F3GA-agarose, and (d) polyuridylic acid-agarose. This heterogeneity was completely eliminated when tunicamycin (2 microgram/ml) was present during induction of interferon; all activity was then recovered in the breakthrough fractions from all sorbents. The level of interferon activity in leukocyte culture fluid was not affected by tunicamycin within the range of concentration 0.05 to 2.0 microgram/ml. These data indicate that (a) human immune interferon undergoes glycosylation, and tunicamycin is an effective inhibitor of this process. Thus, it appears that (b) at least some of the carbohydrates of human immune interferon are N-glycosidically linked. Moreover, it seems that (c) glycosylation is not necessary for an interferon molecule to either be secreted by the cell or (d) to express its antiviral function. Such properties of human immune interferon as (e) the apparent hydrophobicity and (f) an affinity for a polyribonucleotide are conferred only when its glycosylation is unimpaired.
The synthetic interferon (IF) inducer rIn.rCn was modified by substituting the ribosyl residues with either their corresponding deoxy(dC or dI) or 2'-O-methyl analogues (mC or mI). The polynucleotide duplexes of these analogues are inactive as IF inducers. Circular dichroism (CD) studies revealed that, while the deoxy substitution produces significant changes in the conformation of rIn.rCn, the 2'-O-methyl substitution produces no detectable change. Biological competition experiments indicated that the methylated duplexes mIn.rCn, rIn.mCn, and mIn.mCn all compete with rIn.rCn for IF induction, while the deoxy duplexes dIn.rCn and rIn.dCn do not. These results are consistent with those predicted from the CD data. Copolymer duplexes (mI,rI)n.(mC,rC)n of varying degrees of methylation and residue clustering were also evaluated for IF induction in human fibroblasts. The IF-inducing capabilities of these duplexes correlated highly with the presence of clusters containing six or more consecutive ribosyl residues. These combined observations suggest that interaction of rIn.rCn with the cell in the induction process may occur in a biphasic manner involving first the topological recognition of a large segment of the RNA to allow for proper binding to the putative cellular receptor, followed by recognition of a much smaller region of the RNA corresponding to 6--12 consecutive ribosyl residues (0.5 to 1 helical turn) which is responsible for the triggering of the induction process.
Molecular sieving of human leukocyte interferon revealed an apparent molecular weight of 26,000. However, after denaturation by guanidine hydrochloride in the presence of a reducing agent and reactivation by extensive dialysis, a molecular weight of only 21,000 was observed. The reactivated human leukocyte interferon (mol wt 21,000) gave a single peak of activity when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, confirming that a single molecular weight species was generated by the denaturation and reactivation procedure. A partial unfolding of the molecule was evident when the interferon preparation was heated to 50 degrees C in the absence or presence of an unfolding agent and then sieved on Sephadex G-100 Superfine. These results suggest that the interferon molecule undergoes a proteolytic cleavage probably by a protease present in extracellular fluid. Thus, a peptide fragment dissociates from the parent molecule when human leukocyte interferon is denatured in the presence of a reducing agent, resulting in a drop of 5,000 in molecular weight; interestingly, the resultant 21,000 molecular weight form still retains its antiviral activity.
Human fibroblast interferon, obtained by chromatography on concanavalin A-agarose, was stable for at least a month in 30--50 per cent ethylene glycol at 4 degrees, --20 degrees, and --70 degrees C. The succinct point of the present finding is that human fibroblast interferon may be stabilized by ethylene glycol alone without the addition of bovine serum albumin and 'back-contamination' of the interferon preparation.
The antiproliferative effect of mouse L-cell interferon, purified by bovine serum albumin--agarose chromatography (which yields a sp act of 3 X 10(8) international reference U/ml protein), and its two subcomponents, obtained by CH-Sepharose 4B chromatography, has been studied in Balb/3T3 fibroblast cultues. Four to 10 international reference U of interferon/ml resulted in a 50% inhibition of DNA synthesis and cell growth, as determined by the incorporation of [3H]thymidine and cell counts, respectively. Hence the degree of inhibition produced by the purified interferon preparations was comparable to that produced by the original crude interferon (sp act, 1.5 X 10(6) international reference U/mg protein). Our results, obtained with L-cell mouse interferon purified by two novel affinity ligands, provided further evidence linking the antiviral and antiproliferative activities of this molecule.
A series of structurally related ansamycins have been analyzed, in a new immobilized template assay, to determine the mechanism by which they inhibit a ribonucleic acid-directed deoxyribonucleic acid (DNA) polymerase from Moloney murine leukemia virus. By this assay, we can better correlate specific structures of these drugs with inhibitory mechanisms. Using an immobilized template, we were also able to observe drug effects on the stability of complexes formed between the polymerase, a template (polyadenylic acid-agarose), and a primer, as well as to monitor the synthesis of DNA in the presence of drug. For each drug, we determined the complex (intermediate in DNA synthesis) which was primarily affected and whether the effect was due to a destabilization process. Although the activity and specificity of the unsubstituted ansamycins (streptovaricins and rifamycin SV) were modulated by conformation of the molecule and electron density of the aromatic ring, the principal mode of inhibition is, apparently, drug binding to a polymerase-template complex; the drug binds in a manner which prevents subsequent formation of a polymerase-template-primer complex. However, some derivatives of rifamycin SV, when substituted at carbon-3 with bulky or hydrophobic side chains, displayed markedly different modes of action. For example, demethyl dimethyl rifampin prevented the formation of polymerase-template complexes, whereas rifazacyclo 16 acted by promoting the dissociation of polymerase-template-primer complexes.
To develop resources for large-scale production of human fibroblast interferon, we isolated, cryopreserved, and characterized 15 new strains of human diploid foreskin fibroblasts. Their life spans in vitro ranged from 52 to 72 population doublings. We based the selection of cell strains for mass interferon production on the number of population doublings during which consistently high yields of interferon were obtained after "superinduction" in roller bottles; our data show that aging in vitro leads to significant decline in amounts of interferon produced. In contrast, susceptibility to interferon remains largely unaffected by in vitro senescence. Karyotypic analysis indicated that the best interferon-producing strain, MLD (over 60,000 reference units/ml), has a translocation between chromosomes 5 and 15.