Independent sensitivity of human tumor cell lines to interferon and double-stranded RNA.

The antiproliferative effect of human interferons (IFNs) and double-stranded RNAs (dsRNAs) was measured in eight human tumor cell lines, five of which were derived from carcinomas of the bladder. Dose-response curves were generated for a 72-hr treatment period. The concentration of interferon or dsRNA necessary to inhibit tumor cell growth 50% compared to untreated cells was generated by linear regression analysis of the dose-response data. In the seven of eight cell lines in which a direct comparison could be made, IFN-beta was a more potent inhibitor than IFN-alpha. Polyriboinosinic acid X polyribocytidylic acid consistently gave an increased antiproliferative response compared to its mismatched analogue, rln X r(C12,U)n. Correlations could not be made between either IFN-alpha or IFN-beta and the dsRNA effect. No correlation was seen between IFN or dsRNA sensitivity and cell type, ability to bind IFN, growth rate, or tumorigenicity in nude mice. The antiproliferative effect of dsRNA was studied in the presence of antibodies against IFN-beta in HT1080 Cl 4, a cell line sensitive to both IFN and dsRNA, and A2182, a cell line relatively resistant to IFN-beta but sensitive to dsRNA. In both cell lines, the anti-IFN-beta antibodies inhibited the antiproliferative effect of the dsRNAs. After treatment with a concentration of dsRNA necessary to inhibit tumor cell growth 50% compared to untreated cells, a concentration of IFN-beta necessary to inhibit tumor cell growth 50% was induced in the HT1080 Cl 4 cells; however, only a low level of IFN-beta was detected in the culture medium of the A2182 cells.

Antiproliferative and immunomodulatory actions of beta-interferon and double-stranded RNA, individually and in combination, on human bladder tumor xenografts in nude mice.

A cell line, RT4, derived from a human transitional cell carcinoma of the bladder, was grown as a xenograft in athymic mice. The growth of the xenografts was inhibited by beta-interferon (IFN-beta), polyriboinosinic acid.polyribocytidilic acid, the mismatched double-stranded RNA analogue r(I)n . r(C12,U)n, and to a lesser extent recombinant IFN-beta, when treatment was initiated at the time of tumor inoculation. In contrast, the growth rate of established tumors, approximately 6 mm in diameter at the initiation of therapy, was inhibited by both double-stranded RNAs, but not natural IFN-beta, indicating a possible tumor size dependence on the effectiveness of IFN-beta. Combinations of natural or recombinant IFN-beta with either polyriboinosinic acid.polyribocytidilic acid or r(I)n.r(C12,U)n gave an antagonistic effect regardless of tumor mass at the initiation of treatment. This antagonism could be overcome by alternating r(I)n. r(C12,U)n and natural IFN-beta treatment. Natural killer cell activity against RT4 cells in culture was augmented in the spleens of mice treated with r(I)n.r(C12,U)n, but not in those treated with natural IFN-beta. RT4 cells treated in culture with IFN-beta, however, were significantly less efficient as targets for natural killer cells from r(I)n.r(C12,U)n-treated and control spleens. These results indicate that: the effectiveness of IFN-beta may be related to the tumor mass; double-stranded RNAs appear to work, at least partially, in an indirect, immunomodulatory manner; combination therapy can yield an antagonistic rather than an additive or synergistic antitumor effect; and strategic scheduling can overcome the antagonistic effect of combination therapy.

Differential expression of the globin genes in human leukemia K562(S) cells induced to differentiate by hemin or butyric acid.

Human leukemia K562(S) cells were induced to differentiate by 50 microM hemin or 1.4 mM butyric acid, and the types of hemoglobins synthesized were compared. In both cases, embryonal hemoglobins [Portland, Gower 1, Hb X, and fetal hemoglobin (Hb-F)] were detected. Butyric acid-treated K562(S) cells contained mostly Hb Gower 1 (zeta 2 epsilon 2) and a hemoglobin with the electrophoretic characteristics of Portland (gamma 2 zeta 2). For hemin-treated K562(S), the most abundant hemoglobin synthesized by Hb X (epsilon 2 gamma 2), and the second most abundant was Bart's (gamma 4). Traces of Gower 1 were observed in nontreated K562(S) cells. The kinetics of hemoglobin induction as a result of the two treatments differed; increased hemoglobin synthesis was detected after only 24 hr of hemin treatment, whereas 4 days were required in butyric acid-treated cells. Both hemin and butyric acid were able to induce their respective patterns of hemoglobin synthesis independent of the presence of serum in the K562(S) growth medium. Analysis of the globin chains in induced K562(S) cells induced to differentiate indicated that, with both inducers, adult alpha- but not beta-globin chains were present. Karyotype analysis of K562(S) cells revealed a nearly triploid chromosome complement with a modal number of 68 chromosomes. Three copies of chromosome 11 and four copies of chromosome 16 (coding for the beta-like and alpha-like globin genes, respectively) were present. A large marked chromosome, involving chromosome 7, and a Philadelphia chromosome were also seen. These data characterize the K562(S) subline and also indicate that hemin and butyric acid differ in their effects on the expression of embryonal globin genes.

Cloning of a non-c-myc DNA fragment from the double minutes of a human colon carcinoid cell line.

The cell line COLO 320 DM, derived from an untreated human colon carcinoid tumor, was subcloned to obtain a population (Cl 11) with an average of 37 double minutes (DM) per cell. Fractionation of the chromosomes by differential centrifugation yielded a fraction enriched in DM. DNA isolated from the DM-enriched fraction was inserted into the Pst I site of pBR322. One clone, p446, representative of a number of similar clones, contained a region complementary to genomic unique sequences (region p446U). Southern blot analysis using COLO 320 DNA, and DNA from two other cell lines derived from the same biopsy, COLO 320 HSR and COLO 321 HSR, demonstrated amplification and rearrangement of sequences complementary to p446U when compared with 28 different tumor and normal cell lines, some of which contained DM or homogeneously staining regions (HSR). COLO 320 DM Cl 11 had approximately 110 copies per cell of the p446U sequence, or three copies per DM. COLO 320 HSR, which contained one HSR, had 35 copies per cell, while COLO 321 HSR, which contained two HSR, had 700 copies. In addition, p446U did not hybridize with insert sequences of recombinant plasmid pHM(E + H), which includes the human c-myc coding region, 3 kb of upstream flanking sequences and 0.5 kb of downstream flanking sequences, or with an exon 3 probe, pMYC RI-CLA. Amplification of p446U was also not seen in cell lines containing amplified c-myc or N-myc genes. These results indicate that more than one sequence may be amplified in DM or HSR containing tumor cells, but that they need not be amplified together in other tumors.

Implications of retroviral and oncogene activity in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a stem cell disease which, on a clinical level, progresses from the release from growth control of normally differentiated cells (a preleukemic state) to an acute leukemia. On a molecular level, the evolution of CML to acute leukemia is a multistep process. We propose that an early step, at the stem cell level, is acquisition of the ability for gene movement, which allows subsequent submicroscopic and chromosomal rearrangements that cause changes in the growth characteristics and regulation of the stem cell. A specific platelet DNA polymerase (PDP - reverse transcriptase) may play a role in gene movement. The characteristic reciprocal translocation of chromosomes #9 and #22, causing the activation of the c-abl oncogene, appears to be responsible for the uncontrolled cellular growth. Yet, other growth factors (e.g., platelet derived growth factor) and activated oncogenes (e.g., c-sis) must be responsible for the stimulation, progression, and variability seen during the course of the disease. Because CML is a progressive disease with clinically definable stages, CML appears to be a model system for the study of the molecular basis of the progression of preleukemia to leukemia specifically, and preneoplasia to aggressive neoplasia in general.

RNase L and increased endoribonuclease activities in the mononuclear cells of patients with chronic myelogenous leukemia.

During investigations of the interferon-induced 2',5' oligoadenylate synthetase/RNase L system in malignancy, RNase L activity and an increased endoribonuclease activity were observed in peripheral blood mononuclear cell (PBMC) extracts from patients with chronic myelogenous leukemia. The cleavage of rRNA from intact ribosomes was used as the assay for both RNase L and the increased endoribonuclease activities. Novel rRNA cleavage products (NCP) were generated by extracts of Ficoll-purified mononuclear cells from chronic myelogenous leukemia (CML) patients and in the granulocytic fraction of both patients and healthy controls. Determination of the time course of rRNA degradation demonstrated that the novel cleavage products were rapidly derived from the further endoribonucleolytic degradation of the RNase L derived specific cleavage products. Prolonged incubation of mononuclear cell extracts from healthy controls also yielded the novel rRNA cleavage products. Comparisons of the kinetics of NCP production suggest that the novel endoribonuclease activity can be approximately 240-fold greater in PBMC extracts from CML patients than controls. Analysis of peripheral blood WBC count and differential indicated that the increased RNase activities were associated with the presence of immature granulocytic cells in the peripheral blood (p = 0.001, Fisher's exact test). However, these activities were also found in the mononuclear cells of a CML patient in lymphoid blast crisis. Since CML is a stem cell disease, the novel endoribonuclease activity may be indicative of active disease, rather than a marker for immature granulocytes. Thus, the RNase L and increased endoribonuclease activities may play a functional role in the biology of chronic myelogenous leukemia and may be important in the mechanism of action of interferon therapy in this disease.

Heterogeneous nuclear RNA from hairy cell leukemia patients activates 2',5'-oligoadenylate synthetase.

Interferon treatment of cells induces double-stranded RNA (dsRNA)-dependent 2',5' oligoadenylate (2-5A) synthetase, an enzyme which has been implicated in the mechanism of growth arrest in tumour cells. Since interferon (IFN) can inhibit the growth of cells that are not infected with virus, natural non-viral dsRNAs should be present in these cells which can activate 2-5A synthetase. If such nuclear dsRNAs are associated with the mechanism of growth control, cells inherently sensitive to growth inhibition by IFN should contain significant levels of 2-5A synthetase-activating dsRNAs. We measured the ability of size fractionated nuclear dsRNAs isolated from patients with hairy cell leukemia (HCL) to activate purified 2-5A synthetase. Peripheral blood mononuclear cells from HCL patients were utilized because of the inherent sensitivity of these patients to IFN treatment. The heterogeneous nuclear RNA fraction from four out of five HCL patients showed high levels of 2-5A synthetase-activating dsRNAs. The 2-5A formed contained biologically active trimers, tetramers, pentamers and hexamers as demonstrated by HPLC analysis and their ability to activate RNase L. In contrast, the nuclear RNA fraction from three out of four healthy controls were unable to activate 2-5A synthetase. These results indicate that natural, nuclear dsRNAs inherently exist in IFN-sensitive cells and imply that these molecules may play a role in the inhibition of cellular growth.

Differential effects of human natural interferon-alpha and mismatched double-stranded RNA against a human renal cell carcinoma xenograft.

The antitumor effects of natural human IFN-alpha and mismatched dsRNA against the human renal cell carcinoma cell line 786-0 were studied both in a clonogenic soft agar assay and in the nude mouse. The 786-0 cells were sensitive in vitro to the antiproliferative effects of IFN-alpha in a dose-response manner, up to 3000 IRU/ml. These cells were also sensitive, in a dose-dependent manner, to mismatched dsRNA in the clonogenic assay. Mismatched dsRNA was effective in inhibiting tumor growth (p less than 0.001) in nude mouse xenografts, with regression of the tumor mass seen in all animals. A significant increase in survival (p less than 0.001) was seen in the mismatched dsRNA treated group. In contrast, IFN-alpha did not inhibit tumor growth in vivo, even though significant titers of IFN-alpha (greater than 3,000 IRU/ml) were found in the serum shortly after treatment. Mismatched dsRNA did not induce the production of human IFNs by the tumor cells in vitro. Assays of mouse IFN induction and their in vitro antigrowth effects indicated that the in vivo antiproliferative effect of mismatched dsRNA was probably not due to potentiation of any direct effects by the induced mouse IFNs. Tumor growth inhibition appeared to occur, at least in part, from the significant augmentation (p less than 0.01) of natural killer cell activity by mismatched dsRNA, as measured in the spleen cells of treated mice. These results suggest that, although both IFN-alpha and mismatched dsRNA can be directly antiproliferative against this tumor, either the IFN-independent antitumor effects of mismatched dsRNA or the mismatched dsRNA-induced augmentation of the host immune response plays a major role in tumor regression. Potentially, both mechanisms may be important in this system.

Synergistic inhibition of AZT-resistant HIV by AZT combined with poly(I):poly(C12U), without synergistic toxicity to bone marrow progenitor cell elements.

Mutation of human immunodeficiency virus (HIV) to drug resistance is an obstacle to HIV containment, and may account for the transitory nature of the improvement in CD4 cell counts of patients receiving azidothymidine (AZT). The emergence of AZT-resistant (AZTR) virus might be suppressed if a second therapeutic could be added; however, such a regimen would have to confer not only additional control over HIV replication but also no additional toxicity, especially to bone marrow progenitor cells. In the present study, HIV was isolated from patients receiving AZT alone and was studied for sensitivity to the mismatched double-stranded RNA, poly(I):poly(C12U) (ampligen). In addition, the combination of poly(I):poly(C12U) plus AZT was studied in vitro for toxicity to bone marrow CFU-GM and in patients receiving combined therapy for bone marrow toxicity. HIV isolated from patients receiving AZT alone showed higher resistance to AZT than wildtype virus, but remained sensitive to poly(I):poly(C12U). Poly(I):poly(C12U) and AZT were synergistic in inhibiting all isolates of HIV tested, regardless of their AZTR phenotype. Furthermore, the combination of poly(I):poly(C12U) and AZT showed no toxicity in vitro to bone marrow CFU-GM compared to AZT alone. In 11 HIV infected individuals receiving the combinational regimen, bone marrow function gradually improved. These results indicate that poly(I):poly(C12U) was active against AZTR HIV, synergistic with AZT and did not convey added toxicity.

Synergistic antiproliferative effect of human interferons in combination with mismatched double-stranded RNA on human tumor cells.

Four human tumor cell lines were studied for their response to antiproliferative effects of various interferons (IFNs) alone and in combination with the novel mismatched dsRNA, r(I)n r(C12,U)n (Ampligen). RT4 cells (bladder carcinoma) were resistant to Ampligen alone, while A2182 (lung carcinoma), HT 1080 C14 (fibrosarcoma) and RT112 (bladder carcinoma) cells were inhibited in a dose-dependent manner. In contrast, RT4 cells were sensitive to the antitumor effects of IFNs as were HT1080 C14 and RT112 cells, while A2182 cells were resistant. In 3 of 4 cell lines, the recombinant IFNs were less effective than the corresponding natural IFNs when compared by analysis of variance on an IRU/ml basis over a range of concentrations. In all cell lines, a synergistic antiproliferative effect was seen with all IFN preparations studied in combination with Ampligen, as calculated by the isobole method according to Berenbaum (1981). The antiproliferative effect of IFN was potentiated greater than 3.3- to greater than 250-fold, depending on the cell lines, IFN, and concentrations used. Varying the concentration of beta ser-IFN while holding the Ampligen concentration constant gave synergy at all of the physiologically achievable concentrations tested in RT4 cells. These results indicate that: Ampligen worked synergistically with all IFNs in all cell lines studied; growth inhibition of cells resistant to IFNs can be potentiated by low doses of Ampligen; the antiproliferative effect of IFNs can be potentiated by Ampligen in Ampligen-resistant cells; and Ampligen may work by a mechanism other than, or in addition to, the induction of IFNs.

Silver staining as an indicator of active ribosomal genes.

Silver nitrate has been used as a cytological stain since the late 1800s. A modification of the Bielschowsky technique preferentially stains nucleoli and chromosomal nucleolus organizer regions (NORs). The specificity of staining is related to the method of preparation of the cytological specimens. The silver binds proteins and may be associated with the phosphate groups of certain phosphoproteins. Biochemical analyses of nucleolar proteins indicate that a limited array of specific proteins bind silver. A number of investigations have demonstrated that silver staining is indicative of active ribosomal RNA transcription, although a minor component may be associated with the fibrillar centers of cells in which ribosomal genes are inactive. Silver staining is a simple, reliable cytological method for the demonstration of ribosomal gene activity.

Sensitivities of human glioma cell lines to interferons and double-stranded RNAs individually and in synergistic combinations.

The antiproliferative effects of human interferons (IFNs) and double-stranded RNAs (dsRNAs) were studied in five human glioma cell lines. Dose response curves were generated over a 72 hour treatment period. The concentration of interferon or double-stranded RNA necessary to produce a 50% antiproliferative response (GI50) was calculated by linear regression analysis. Two cell lines were more sensitive to IFN-beta than to IFN-alpha, one cell line was more sensitive to IFN-alpha than to IFN-beta and two cell lines had approximately equal sensitivities to both interferons. All cell lines showed some sensitivity to either IFN-alpha or IFN-beta. IFN-gamma had no antiproliferative effect on any of the cell lines. In addition, only one of the cell lines displayed sensitivity to dsRNA, in which the response to poly(I).poly(C) was greater than that to a mismatched analogue of poly(I).poly(C), r(I)n.r(C12,U)n (Ampligen). There was no correlation between the sensitivities to type I IFNs (alpha and beta), type II IFN (gamma) or the dsRNAs. The antiproliferative effect of combinations of IFNs, or IFNs and Ampligen, was studied in one of the cell lines. A significant synergistic antitumor effect was seen with all of the IFN/Ampligen combinations (p less than 0.02), including IFN-gamma/Ampligen, even though these cells were resistant to IFN-gamma alone. Synergy was also seen in the IFN-alpha/IFN-gamma (p less than 0.02) and IFN-beta/IFN-gamma (p less than 0.05) combinations. The IFN-alpha/IFN-beta combination gave an additive antitumor effect. These results indicate that IFN-alpha and IFN-beta alone or combinations of type I IFNs, type II IFNs and Ampligen can be effective in inhibiting the growth of glioma cells.

Identification of nucleolus organizer regions (NORs) in normal and neoplastic human cells by the silver-staining technique.

Silver nitrate has been used to demonstrate the chromosomal location of ribosomal cistrons in nine tissue-culture lines derived from human tumors of various pathological origins. Control individuals have a particular modal number (range 7--10) of D- and G-group chromosomes stained with silver. In the controls, 96.2% of the D- and G-group chromosomes that have a stalk show silver staining, while no relationship can be seen in acrocentric chromosomes without stalks. The tumor cells, whose modal chromosome numbers range from 42 to 68, possess variable numbers of acrocentrics (11--18). The number of chromosomes stained with silver, however, remained at control levels (range, 6--9). These data indicate that, in humans, silver staining may not identify all NORs that contain structural ribosomal genes.

Potentiated lymphokine-activated killer cell activity generated by low-dose interleukin-2 and mismatched double-stranded RNA.

Lymphokine-activated killer (LAK) cell activity was measured in human peripheral blood mononuclear cells (PBMC) treated in vitro for 3 days with recombinant interleukin-2 (rIL-2) and mismatched double-stranded RNA (dsRNA). Lytic activity was measured utilizing K562 (NK-sensitive) and 786-0 (NK-resistant) target cells. PBMC cultured with rIL-2 (10-1000 BRMP U/ml) alone showed concentration-dependent lytic activity against the 786-0 target cells, while cells cultured in unsupplemented medium or medium supplemented with mismatched dsRNA (200 micrograms/ml) alone could not lyse the 786-0 targets. The combination of mismatched dsRNA with suboptimal concentrations of rIL-2 (10-30 U/ml) showed enhancement of both natural killer (NK) and LAK cell activities. The uptake of [3H]thymidine by treated effector cells was dependent on time and rIL-2 concentration and was not increased in the cells treated with low-dose rIL-2/mismatched dsRNA, compared to those treated with low-dose rIL-2 or mismatched dsRNA alone. Similarly, changes in the expression of CD3, CD4, CD8, CD57, CD16 and CD25 cell surface antigens were independent or rIL-2 concentration and not altered by the presence of mismatched dsRNA. These results indicate that mismatched dsRNA can potentiate rIL-2-induced LAK cell activity by increasing the functional activity per cell, rather than by increasing the number of activated cells.

Biological activity of mRNA immobilized on nitrocellulose in NaI.

In 12.2 molal NaI and at 25 degrees C or below, mRNA bound to nitrocellulose while DNA and rRNA did not. Neither the poly(A) tract nor the cap were required for binding. The immobilized RNA could be translated, reverse transcribed, hybridized with radioactive probes, or released for further manipulation. mRNA was efficiently transferred from polyacrylamide to nitrocellulose in NaI. Baking was not required to fix NaI-immobilized mRNA to nitrocellulose. When cells dissolved in 12.2 molal NaI were filtered through nitrocellulose, mRNA became selectively bound (quickblot). The quick-blot system utilizing protease and detergents to prepare cells for NaI solubilization was especially suitable in quantitative, rapid screening of cells for expression of specific genes. Expression of highly repeated DNA sequences was detected in human leukemia cells.

S1 nuclease definition of highly repeated DNA sequences in the Guinea pig, Cavia porcellus.

Native DNA of the Guinea pig, Cavia porcellus, purified from liver or tissue culture cells, was heat denatured and reassociated to a Cot value of 0.01 (equivalent Cot value of 7.2 x 10(-2)). The reassociated DNA was isolated by digestion with the single-strand DNA specific enzyme S1 nuclease. Spectrophotometric and radioactivity assays demonstrated that 24% of the total DNA was resistant to S1 nuclease treatment. Zero-time reassociation indicated that approximately 3% of the DNA was inverted repeat sequences. Thus, highly repeated sequences comprised 21% of the total genome. CsCl buoyant density ultracentrifugation indicated that this fraction was composed of both main band and satellite sequences. Although actinomycin D - CsCl density gradients failed to give significant separation of the repetitive sequences, distamycin A - CsCl gradients were able to fractionate the DNA into several overlapping bands. The heterogeneity of the repetitive DNA was further demonstrated by the first derivative plots calculated from their thermal denaturation profiles. This analysis revealed six major thermalytes which indicate that there may be at least six discrete components in the repetitive DNA.

Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions.

Nucleoli isolated from Novikoff hepatoma cells were stained with AgNO3 to demonstrate the typical staining of active ribosomal cistrons. Pre-treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 2.0 M NaCl did not interfere with silver staining. Treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 0.15 M NaCl did, however, eliminate silver binding. Serial extraction of nucleoli with 2.0 M NaCl buffer followed by 0.15 M NaCl buffer also abolished silver staining. Analysis of the supernatant fraction of these extracts by polyacrylamide gel electrophoresis indicates that, although more than one nucleolar protein can bind silver, only one protein is associated with the staining of active ribosomal cistrons.

Differential antiproliferative actions of 2',5' oligo A trimer core and its cordycepin analogue on human tumor cells.

The antiproliferative effect of 2',5' A3 core and 2',5'-3'dA3 (cordycepin trimer) core was measured in 8 human tumor cell lines. Cells were treated in a dose-response manner for 72 hr and the concentration of drug necessary to inhibit cell growth 50% (GI50) was determined. A wide range of sensitivities to these drugs was found, even among tumors of the same histological type. The cell lines showed different sensitivities and dose-response curves to the 2',5'A3 and 2',5'-3'dA3 cores. Uptake studies of the 2',5'A3 and 2',5'-3'dA3 cores, using high-pressure liquid chromatography, demonstrated that both cores were rapidly degraded in the tissue culture medium and taken up as adenosine or cordycepin, respectively. There was a direct correlation between the uptake of cordycepin and the antiproliferative effect. In contrast, there was no correlation between cell sensitivity and the uptake of the 2',5'A3 core degradation products. Analysis of intracellular nucleosides and nucleotides indicated that differences in intracellular metabolism of adenosine might explain the different sensitivities of the various cell lines to 2',5'A3 core. Molar equivalent concentrations of adenosine and cordycepin inhibited cell growth; however, equimolar concentrations of these nucleosides were not effective. In addition, the antiproliferative effect of both core compounds and their corresponding nucleosides could be potentiated by the addition of the adenosine deaminase inhibitor, deoxycoformycin. The results indicate that these cores act as prodrugs and that the active metabolites are their corresponding nucleosides.

Preclinical studies with Ampligen (mismatched double-stranded RNA).

Historically, double-stranded (ds) RNAs have been largely over-looked as potentially valuable anticancer/antiviral drugs, primarily because of the many clinical toxicities and lack of efficacy associated with the first clinically tested dsRNA--polyinosinic-polycytidylic acid (rIn X rCn). However, studies summarized herein demonstrate that the therapeutic ratio of dsRNAs can be greatly enhanced by purposeful mispairing of bases. For example, a mispaired dsRNA, termed Ampligen (rIn X r(C12,U)n), shows strong antitumor activity in a variety of relevant test systems with little or none of the toxicities associated with rIn X rCn. Furthermore, Ampligen demonstrates a much wider therapeutic spectrum than that displayed to date by any single type of interferon (natural or recombinant DNA-derived). Importantly, Ampligen, the product of a straight-forward enzymatic synthesis, shows excellent lot-to-lot biological and biophysical specifications, which is often not the case with biologically derived new compounds. Furthermore, a significant fraction of human solid tumors, which are largely unresponsive to conventional chemotherapy or interferon (IFN), is sensitive to Ampligen in a fresh human tumor clonogenic assay. Indeed, whereas 50% of untreated and IFN-treated athymic mice engrafted with human renal cancer cells die within 20-22 weeks, mice treated with Ampligen survive a minimum of 32 weeks (p less than 0.0003). A summary of all animal models tested and human clinical trials to date demonstrates that Ampligen exerts a greater antitumor activity than IFN and has a superior therapeutic ratio compared to rIn X rCn.