Dose-related effects of Ampligen (poly(I).poly(C12U)), a mismatched double-stranded RNA, in a bleomycin-mouse model of pulmonary fibrosis.

The antifibrotic effect of the mismatched double-stranded RNA, Ampligen (poly(I).poly(C12U)), was evaluated in a bleomycin-mouse model of pulmonary fibrosis. Mice received a single intratracheal dose of bleomycin (0.125 U/mouse) or saline (50 microL) at the beginning of the experiment, followed by 5 or 6 intraperitoneal injections of Ampligen (1.0, 5.0, 10.0, 15.0, or 25.0 mg/kg) or saline at regular intervals for 2 weeks. Ampligen did not produce increased mortality or weight loss by itself. However, it produced varying degrees of mortality in combination with bleomycin. Five injections of 10 mg/kg Ampligen or three injections of 25 mg/kg Ampligen plus three injections of 10 mg/kg Ampligen in combination with bleomycin .produced significant reductions in lung collagen accumulation as indicated by lung hydroxyproline content compared to the bleomycin control group. Animals receiving bleomycin plus Ampligen at all dosages had significantly reduced prolyl hydroxylase activity compared to the bleomycin control group. Lipid peroxidation and bronchoalveolar lavage fluid (BALF)-supernatant protein content for the groups receiving bleomycin plus Ampligen were not reduced compared to the bleomycin control group. In the BALF-supernatant, the activity of acid phosphatase, a lysosomal enzyme produced by neutrophils, monocytes, and macrophages, was significantly decreased in the group receiving bleomycin plus 10 mg/kg Ampligen. Also, selected BALF differential immune cell counts were reduced in some of the groups receiving bleomycin plus Ampligen, but not in a consistent or dose-dependent manner. The results of this study indicate that Ampligen can significantly reduce the bleomycin-induced increased collagen accumulation and may be therapeutically useful in the management of lung fibrosis in humans.

A 2',5'-oligoadenylate analogue inhibits murine hepatitis virus strain 3 (MHV-3) replication in vitro but does not reduce MHV-3-related mortality or induction of procoagulant activity in susceptible mice.

Exposure of inbred mice to murine hepatitis virus strain 3 (MHV-3) causes a strain dependent spectrum of disease symptoms which correlates with induction of procoagulant activity (PCA) by macrophages. Previous studies have demonstrated a role for interferons in resistance to MHV-3 infection. These cytokines have both antiviral and immunoregulatory effects which may be crucial for MHV-3 resistance. One of their antiviral effects is the ability to induce 2',5'-oligoadenylate (2-5A) synthetase leading to activation of the latent endoribonuclease RNase L. Once activated, RNase L degrades ssRNA thereby inhibiting viral-induced protein synthesis. These studies were undertaken to determine the effects of Oragen 0004 (Oragen), an RNase L activating 2-5A analogue, on MHV-3 replication and induction of PCA in vitro and on the course of MHV-3 infection in susceptible BALB/cJ mice in vivo. Oragen inhibited MHV-3 replication in peritoneal macrophages derived from resistant A/J and susceptible BALB/cJ mice in a dose-dependent fashion. Concentrations of Oragen greater than 110 micrograms/2 x 10(6) macrophages decreased viral replication by greater than 89% in peritoneal macrophages in vitro obtained from both BALB/cJ and A/J mice and by 86% in livers from MHV-3-infected mice in vivo. However, Oragen failed to inhibit induction of PCA following in vitro exposure of BALB/cJ mice-derived peritoneal macrophages to MHV-3 and failed to prevent the development of fulminant hepatitis in BALB/cJ mice in vivo. Thus, these studies demonstrate clearly that induction of 2-5A synthase and inhibition of viral replication is not sufficient to prevent MHV-3-related hepatocellular injury, and these data further support the role of PCA in the pathogenesis of MHV-3 infection.

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.

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.

Cellular and enzymatic activities of a synthetic heteropolymer double-stranded RNA of defined size.

We have synthesized a novel heteropolymer double-stranded RNA (dsRNA) molecule of defined length and strandedness (dsRNA309) and evaluated its ability to induce cytokine gene expression, activate dsRNA-dependent enzymes, and inhibit both tumor cell growth and virus replication. Unlike the conventionally studied synthetic homopolymer dsRNAs, polyinosinic acid:polycytidylic acid (poly(I-C)) and its mismatched analogue polyinosinic:polycytidylic, uridylic acid (poly(I-C12,U), dsRNA309 possessed restricted biological activity. dsRNA309 was unable to inhibit tumor cell growth or efficiently induce cytokine (i.e. interferon-beta and interleukin-1 alpha) gene expression. However, dsRNA309 was able to inhibit virus replication and activate dsRNA-dependent intracellular enzymes, 2'-5' oligoadenylate synthetase (2'-5' A synthetase) and the dsRNA-activated inhibitor kinase in in vitro assay systems. Overall, dsRNA309 provided a means for examining the mechanisms governing the dsRNA-regulated antiviral and antiproliferative responses, and studies with dsRNA309 demonstrated that the ability of a synthetic dsRNA to activate dsRNA-dependent intracellular enzymes does not necessarily predict the same gene inducing capacity.

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.

Antitumor effects of interleukin-2 and mismatched double-stranded RNA, individually and in combination, against a human malignant melanoma xenograft.

The antitumor effects of recombinant interleukin-2 (rIL-2) and mismatched double-stranded RNA (dsRNA) were assessed in tissue culture and in a nude mouse model. Mismatched dsRNA did not show a direct antiproliferative effect against the human malignant melanoma cell line, BRO, in tissue culture. However, treatment of the BRO cells with up to 1000 units/ml rIL-2 in culture showed a slight increase in growth rate. Combined rIL-2/mismatched dsRNA treatment also demonstrated a similar slight enhancement of growth. Nude mice bearing subcutaneous tumors were treated by intraperitoneal injection of low doses (5000-20,000 units) of rIL-2 and mismatched dsRNA (500 micrograms). The in vivo tumor growth was significantly inhibited by the combined treatments (P less than 0.05) and survival was significantly increased (P less than 0.05). Measurement of cytotoxicity using splenocytes from treated animals showed significant augmentation of lytic activity against natural killer (NK)-sensitive YAC-1 cells in all rIL-2/mismatched dsRNA treatment groups, compared to the individual treatments or controls (P less than 0.05). Cytotoxicity of the splenocytes against the NK-resistant BRO cells was also augmented in animals treated with mismatched dsRNA and the highest rIL-2 dose utilized here (P less than 0.01). Renal, liver, and hematological toxicity was evaluated by measurement of blood urea nitrogen, creatinine, serum asparrtate aminotransferase, and a complete blood count with differential. There were no significant differences in these parameters in any of the treatment groups. Similarly, no differences in weight of the animals was seen in any treatment group. These results indicate that the combination of low-dose rIL-2 and mismatched dsRNA can potentiate host-mediated antitumor effects, yielding increased survival, without significant toxicity.

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.

Cyclic AMP mediates the direct antiproliferative action of mismatched double-stranded RNA.

Previous experiments have demonstrated that double-stranded RNAs (dsRNAs) can exert an antiproliferative effect on human tumor cells, independent of interferon (IFN) induction. However, the mechanism by which dsRNAs inhibit tumor growth has not been elucidated. As a first step in determining the molecular events responsible for growth arrest, we have explored the role of signal transduction through the cAMP system in the antiproliferative effect of the mismatched dsRNA, r(I)n.r(C12,U)n (Ampligen). These studies utilized the human glioma cell line A1235, which does not produce detectable levels of IFN-alpha, -beta, or -gamma in response to mismatched dsRNA treatment. Treatment of A1235 cells with mismatched dsRNA in combination with either 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), which inhibits cAMP-dependent protein kinase and protein kinase C, or N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), which preferentially inhibits the cAMP-dependent protein kinase, yielded an antagonism of the mismatched dsRNA-induced antiproliferative effect. Measurement of adenylate cyclase activation showed a dose-dependent increase in activity at antiproliferative mismatched dsRNA concentrations, but not at lower, nonantiproliferative doses. This increase in activity was rapid, seen as early as 30 sec after initiation of treatment, and it was sustained at peak levels for 1-2 hr. Analysis of the intracellular cAMP concentration gave similar kinetics of induction. Exposure of cells to the stable cAMP analogue dibutyryl cAMP yielded dose-dependent inhibition of cell growth. The cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also inhibited proliferation. In contrast, neither H-7 nor HA1004 had an effect on growth inhibition induced by human natural IFN-alpha treatment. In addition, antiproliferative doses of IFN-alpha did not increase cAMP concentrations. These results indicate that the cAMP system is utilized by mismatched dsRNA as an early signal transduction mechanism for growth control. Furthermore, the antiproliferative effects induced by mismatched dsRNA and IFN can occur by different mechanisms of action.

Mismatched double-stranded RNA, Ampligen (poly(I): poly(C12U), demonstrates antiviral and immunostimulatory activities in HIV disease.

Mismatched double-stranded RNA (Ampligen) has broad spectrum antiviral and immunomodulatory activities. These activities generate stabilization or improvement in three important surrogate markers of HIV disease progression. Patients with HIV disease treated with Ampligen do not become positive for p24 antigen, in contrast to patients treated with AZT or placebo. Viral burden can also be decreased in patients receiving Ampligen/AZT therapy. In vitro studies indicate that both AZT sensitive and AZT resistant viruses can be inhibited by Ampligen alone and are synergistically inhibited by Ampligen in combination with AZT. The immunomodulatory effects of Ampligen are manifested as a stabilization of CD4 counts. When Ampligen is combined with AZT, an increase in CD4 count is seen. Furthermore, a return or increase in delayed type hypersensitivity to mumps, Candida, and trichophyton was seen in approximately 70% of patients treated with Ampligen. The activity of Ampligen in HIV disease is due to its multifunctional activity as an antiviral and immune stimulating agent. The antiviral effect directly inhibits HIV-infection and other viruses which have been implicated in HIV disease acceleration and progression. The immunomodulatory activity can stabilize, increase, or restore immune function. This enhanced immune function can also lead to the further inhibition of additional infections associated with disease progression. Thus, Ampligen has multiple mechanisms of action against HIV disease.

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.

Augmented antitumor effect of combined human natural interferon-alpha and mismatched double-stranded RNA treatment against a human malignant melanoma xenograft.

The antitumor effect of combined natural human interferon-alpha (IFN) and mismatched double-stranded RNA (dsRNA) treatment against the human malignant melanoma cell line, BRO, was studied. In vitro results, using a tissue culture antiproliferative assay, indicated that these cells were moderately sensitive to IFN-alpha. In contrast, mismatched dsRNA had no antitumor effect, and a minimal stimulation of cell growth, over part of the concentration range tested, was observed. Mismatched dsRNA did not potentiate the antitumor effect of IFN-alpha in cells receiving combination treatment. Xenografts of BRO cells, inoculated subcutaneously into nude mice, were used to evaluate the antitumor effects of IFN-alpha and mismatched dsRNA. Growth of the primary tumor was inhibited by both drugs alone or in combination (p less than 0.001), but the combined treatment was most effective and appeared to be additive. The number of spontaneous lung metastases was also inhibited (p less than 0.02) in all treatment groups. Survival, however, was significantly increased only in the IFN-alpha/mismatched dsRNA group (p less than 0.02 compared to controls, p less than 0.05 compared to mismatched dsRNA alone). Determination of splenic natural killer (NK) cell activity against BRO cells demonstrated that significantly augmented NK activity to the same extent, but that the IFN-alpha alone had no effect. These results indicate that IFN-alpha worked through direct antiproliferative mechanisms while mismatched dsRNA stimulated host immunomodulatory effects. The increased tumor growth inhibition and survival in the dual treatment group appears to result from the combined direct antiproliferative and indirect immunomodulatory effects.

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.

Synergistic antiproliferative effect of recombinant alpha-interferons with recombinant gamma-interferon.

Two human tumor cell lines were studied for their response to the antiproliferative effect of recombinant human interferons (IFNs) alpha 2, alpha 4, a hybrid alpha (delta 4 alpha 2 Bgl II alpha 1), and gamma, individually and in combination. Natural human alpha-IFN was used as a reference point for all experiments. RT4 (bladder carcinoma) cells were overall more sensitive to the antiproliferative effects of the IFNs than A2182 (lung adenocarcinoma) cells. Three-way analysis of variance indicated that the relative effectiveness of the alpha-IFNs was alpha 2 less than alpha 4 less than hybrid alpha less than natural alpha-IFN. On an international reference unit per milliliter basis, gamma-IFN was 50- and 75-fold more effective than natural alpha-IFN and hybrid alpha-IFN in RT4 cells and 5.6-, 12.1-, and 14.9-fold more effective than alpha 4-, hybrid alpha-, and natural alpha-IFN in A2182 cells. In contrast, when recalculated on a nanogram per milliliter basis, gamma-IFN was only threefold more effective than the hybrid alpha-IFN in RT4 and approximately twofold less effective than alpha 4 and the hybrid alpha in A2182. Combinations of alpha-IFNs gave additive or antagonistic effects. When any of the alpha-IFNs were combined with the gamma-IFN, however, a synergistic antiproliferative effect was seen. The magnitude of the synergy was dependent upon the concentration of gamma-IFN used and the type of alpha-IFN in the combination. Antagonistic effects were seen at the lowest gamma-IFN concentration studied (0.2 IRU/ml). Synergy also varied according to the potency of the alpha-IFN used.

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.

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.

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.

Comparative studies of ampligen (mismatched double-stranded RNA) and interferons.

Historically, it has been assumed that double-stranded (ds) RNAs function at a cellular level exclusively via an interferon (IFN) induction mechanism. However, current studies conducted both in the laboratory and at the clinical level reveal that this assumption is incorrect and, indeed, underestimates the intrinsic antitumor activity of certain dsRNAs. A specific dsRNA (Ampligen) shows strong antiproliferative activity against human carcinoid tumor cells in a clonogenic assay when natural alpha- and beta-IFNs were inactive. Similarly, in vivo studies in which human renal cancer cells were transplanted into athymic mice demonstrate a strong antitumor effect of Ampligen whereas such tumors are largely unaffected by alpha-IFN treatment. In a comparative study including many fresh human tumors of various histological types (breast, ovarian, melanoma, renal, and carcinoid) numerous examples were uncovered of Ampligen sensitivity (antiproliferative effect) in the face of relative or complete insensitivity to IFNs. Synergistic effects of Ampligen plus IFN overcame the resistance of some human tumor cells to either biological modifier given alone. It can also be demonstrated that the antitumor action of Ampligen on certain human lung tumor cells is not shared by polyinosinic . polycytidylic acid, thus indicating that different dsRNAs may themselves exhibit dissimilar effects on various human tumors.

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.