Weldon B. Jolley

Roles of interferon and natural killer cells in the antiviral activity of 7-thia-8-oxoguanosine against Semliki Forest virus infections in mice.

7-Thia-8-oxoguanosine is a novel biological response modifier with broad-spectrum antiviral activity against many DNA and RNA viruses in vivo. Since two of its properties are to induce interferon and to activate natural killer (NK) cells, we investigated the roles of the lymphokine and NK cells in the antiviral activity of the compound against Semliki Forest virus. Antibody to interferon alpha/beta could completely abolish the protective activity of the nucleoside against virus infection in mice, whereas antibodies to interferons beta and gamma could not, indicating that interferon alpha was of major importance to confer protection to the animals. Reduced activation of NK cells was also observed in mice treated with 7-thia-8-oxoguanosine and antibody to interferon alpha/beta. The role of NK cells in the protective activity of the compound was directly assessed in beige mice or in Swiss Webster mice treated with asialo GM1 antibody. In both experiments, the animals were protected from lethal virus infection by treatment with nucleoside. Spleen cells primed by 7-thia-8-oxoguanosine and adoptively transferred to untreated mice could not save them from virus-induced mortality. These three results provide evidence that natural killer cells activated by 7-thia-8-oxoguanosine play a minimal role in protection from acute Semliki Forest virus infections in mice.

Activation of the respiratory burst in murine phagocytes by certain guanine ribonucleosides modified at the 7 and 8 positions: possible involvement of a pertussis toxin-sensitive G-protein

The capacity of certain guanine ribonucleosides (modified at the 7 and/or 8 positions) to enhance the respiratory burst of murine peritoneal phagocytes was evaluated. The results show that 8-mercaptoguanosine, 8-bromoguanosine, 7-methyl-8-oxoguanosine and 7-thia-8-oxoguanosine, when injected intraperitoneally into mice, induced peritoneal phagocytes to generate reactive oxygen species as early as 1 h after injection. In vivo administration of the nucleosides induced higher levels of phagocyte activation than in vitro treatment with the same nucleosides. However, the addition of interferon alpha/beta in vitro significantly increased the magnitude of phagocyte activation by the nucleosides, suggesting an important role for cytokines/lymphokines in the nucleoside-induced phagocyte activation in vivo. Furthermore, pre-treatment of phagocytes in vitro with Bordetella pertussis toxin, before treatment with the guanosines, inhibited their capacity to induce the respiratory burst. These observations establish these low-molecular-weight compounds as interesting probes for the study of stimulus-response coupling in phagocytes.

Changes in diacylglycerol and cyclic GMP during the differentiation of human myeloid leukemia K562 cells

When the human myeloid leukemia cell line, K562, was induced to differentiate along the erythroid lineage by a 4 day treatment with 10 microM tiazofurin, the cellular content of diacylglycerol decreased to 35% of the value in untreated control cells. Under the same conditions the content of cGMP decreased to 61% of the control value. Tiazofurin inhibits guanine nucleotide biosynthesis and lowers cellular GTP. When guanosine and adenine were added together with tiazofurin, the differentiation of K562 was prevented, the concentration of diacylglycerol was maintained at control values, and the reduction in the concentration of cGMP was partially prevented. Other inducers of differentiation which acted by different mechanisms, caused similar changes in the concentrations of diacylglycerol and cGMP.

Nucleoside and nucleotide modulation of genetic expression―A new approach to chemotherapy

Unlike conventional enzymes, receptors that activate G proteins do not catalyze the direct formation or cleavage of covalent bonds but act instead as a catalyst for the exchange of GTP vs GDP, which results in major conformational changes in the alpha subunit of G proteins and dissociation and selective binding of the alpha subunit which provokes direct enzyme activation eventually resulting in stimulation of protein kinase A, B or C. Each of these kinases can phosphorylate specific DNA binding proteins which allow new portions of DNA to be read and expressed. Such a series of events can act as switches to control cellular genetic expression resulting in cellular proliferation, differentiation or hormonal secretion of growth factors (Scheme I). Examples of nucleosides and nucleotides which appear to exert their therapeutic effects via G protein control of cellular proliferation resulting in differentiation are tiazofurin, selenazofurin, and 8-chloro-cAMP which have been synthesized and studied in our laboratories. The clinical application of these nucleosides in cancer treatment is presently underway and offers a viable alternative to chemotherapy with highly cytotoxic agents. The use of these derivatives result in down-regulation of the G protein regulatory pathways responsible for rapid cell division. Alternatively, a series of guanosine analogs prepared in our laboratories, 8-bromoguanosine, 8-mercaptoguanosine, 7-methyl-8-oxoguanosine and 7-thia-8-oxoguanosine, all activate various aspects of the immune response by up-regulation of G protein regulatory pathways in various lymphocyte derived cells. Guanosine-like nucleosides which function in this manner could have major clinical application as antitumor, antiviral and antimetastatic agents providing the desired specificity can be achieved. Specific immune enhancement of the aged might be an attainable goal if suitable orally active guanosine derivatives with high specificity can be achieved. The G protein regulatory pathways for modulation of genetic expression in specific cell types provide a major modern approach to new chemotherapeutic agents.

Potentiation of the efficacy of murine L1210 leukemia vaccine by a novel immunostimulator 7-thia-8-oxoguanosine: Increased survival after immunization with vaccine plus 7-thia-8-oxoguanosine

SummaryWe have investigated the ability of a novel immunopotentiator, 7-thia-8-oxoguanosine (7T8OG) to increase the efficacy of a weakly immunogenic murine L1210 leukemia vaccine. The vaccine was prepared by irradiating L1210 leukemia cells in a cesium source with a total of 6000-R dose. DBA/2 mice were treated with 150 mg/kg 7T8OG and/or with vaccine consisting of 107 irradiated cells. In combination therapy, mice first received the vaccine and then were injected with 75 mg/kg 7T8OG 2 h and 4 h after vaccination. One week after the last treatment all mice were inoculated with 104 live leukemia cells intraperitoneally. Control, untreated mice (n = 66) injected with 104 live leukemia cells had a mean survival time ± standard error of 10.5±0.2 days. Treating mice (n = 66) with one, two or three doses of 7T8OG administered i.p. 1 week apart did not increase survival (mean survival time = 10.7 days). Mice immunized with one, two or three doses of vaccine had 14.5±1.1, 45.4±6.2 and 68.3±10.6 days mean survival, respectively. 7T8OG-stimulated vaccination increased the survival dramatically. The best survival was noted when the mice were treated with 2× (vaccine + 7T8OG). Immunization of mice (n = 30) with this treatment regimen increased the mean survival to 156±10.0 days. Over 90% of mice that were treated this way had a cumulative survival time greater than 160 days. In contrast, only 12% of the mice immunized twice with the leukemia vaccine alone survived over 160 days. These results suggest a rationale for the use of this immuno-potentiator with various vaccines for a more effective immunization.

A novel guanosine analog, 7-thia-8-oxoguanosine, enhances macrophage and lymphocyte antibody-dependent cell-mediated cytotoxicity

Intraperitoneal treatment of mice with a novel guanosine analog, 7-thia-8-oxoguanosine (7-thia-8-oxoGuo), gives rise to activated splenic lymphocytes and peritoneal macrophages with enhanced capacity to mediate antibody-dependent cellular cytotoxicity (ADCC). ADCC activities against both chicken red blood cells and P815 murine plasmacytoma cells were enhanced, indicating that macrophages as well as lymphocytes functioning as K-cells in the two distinct cytolytic systems, were activated by 7-thia-8-oxoGuo. Furthermore, 7-thia-8-oxoGuo enhanced lymphocyte-mediated ADCC activity in beige (bgJ/bgJ) mice against P815, thus indicating the ability of 7-thia-8-oxoGuo to function as a potent immunomodulator even in an animal that is known to possess selective impairment of naturally occurring killer lymphocytes. These results suggest that 7-thia-8-oxoGuo could serve as an agent for immunomodulation and immunorestoration.

Inhibition of pyrimidine metabolism in myeloid leukemia cells by triazole and pyrazole nucleosides.

Two triazole nucleosides, 1 (3-beta-D-ribofuranosyl-1,2,4-triazole-5-carboxamide) and 2 (2-beta-D-ribofuranosyl-1,2,3-triazole-4,5-dicarboxamide), and a pyrazole nucleoside, 3 (1-beta-D-ribofuranosylpyrazole-3,4-dicarboxamide), were found to inhibit pyrimidine nucleotide biosynthesis in the human myeloid leukemia cell line, K562. Cells treated with these inhibitors released orotate in quantities of 8-35 nmol/10(5) cells/day. Treatment with these compounds caused the K562 cells to accumulate in the S phase of the cell cycle and induced the cells to synthesize hemoglobin.

Successful immunotherapy of murine melanoma metastases with 7-thia-8-oxoguanosine

We have recently reported that a synthetic nucleoside, 7-thia-8-oxoguanosine (7T8OG) is a potent activator of a number of effectors which are involved in anti-tumor immune responses. 7T8OG was found to induce interferon (IFN) production, to activate asialo-GM1 positive (AGM1+) killer cells, and to enhance specific antibody responses. In the present study, we investigated the effect of 7T8OG on growth of the murine pulmonary B16 melanoma and on formation of metastases. C57BL/6 mice were injected i.p. with 50–150 mg/kg 7T8OG before or after i.v. inoculation of B16 melanoma tumor cells, and 17–19 days after tumor inoculation, the number of metastases in the lungs were counted. 7T8OG given systemically in a single or a divided dose 24 h prior to the challenge of tumor cells reduced the number of lung tumor metastases by 89–99% which is highly significant as compared to untreated control (P<0.001). Occasional extra pulmonary tumor growth in the thoracic cavity and neck lymph node was also completely inhibited. The reduction in the number of tumor nodules was dose dependent. A single dose of 150 mg/kg of 7T8OG was also effective in inhibiting the growth of 3–5 day old metastatic tumors. The cytotoxic activity of killer cells inducedin vivo by 7T8OG was completely abolished byin vitro treatment of cells with antiAGM1 antibody plus complement. Administration of anti-AGM1 antibody following the 7T8OG treatment completely abrogated the anti-tumor effect of 7T8OG, resulting in a massive increase in the number of tumor foci in the lungs. Administration of carageenan or silica followed by injection of 7T8OG caused a significant increase (P<0.01) in the number of pulmonary tumor nodules compared to treatment with 7T8OG only. These findings indicate that activated macrophages or perhaps their cytokine (tumor necrosis factor) also contribute to the host tumor defense by 7T8OG.

Antiviral and Immunoenhancing Properties of 7-Thia-8-Oxoguanosine and Related Guanosine Analogues

7-thia-8-oxoguanosine (TOGuo) is the first reported structure of a family of modified guanosine analogues exhibiting antiviral activity in rodent models. Its spectrum of action includes interferon-sensitive viruses such as alphaviruses, bunyaviruses, corona viruses, flaviviruses, picornaviruses and, to a lesser extent, herpesviruses. Early treatment before or shortly after virus challenge is necessary to protect animals from mortality. The protective effect of TOGuo against Semliki Forest and Punta Toro viruses can be eliminated by co-treatment with antibody to alpha/ beta-interferon. indicating that interferon induction is of prime importance for antiviral activity against these two viruses. Immunological studies indicate that the nucleoside induces alpha-interferon and activates B cells, natural killer cells, antibody-dependent cytotoxic cells and macrophages. TOGuo also has adjuvant activity, since its use in combination with a killed Ll210 leukemia cell vaccine greatly reduced the mortality of mice inoculated with live Ll210 leukemia cells compared with the vaccine used alone. Several related guanosine analogues show similar antiviral and immunoenhancing properties, including 7-methyl-8-oxoguanosine, 7-methyl-8-thioxoguanosine and 7-deazaguanosine. These studies indicate that certain modifications at the 7 and 8 positions of guanosine may confer antiviral and immunostimulatory properties to nucleoside analogues.

Stimulation of phosphoinositide signaling pathway in murine B lymphocytes by a novel guanosine analog, 7-thia-8-oxoguanosine.

A novel guanosine analog, 7-thia-8-oxoguanosine, is shown to induce proliferation in nude mouse splenic lymphocytes in vitro as measured by increased DNA synthesis. Determination of the levels of sn-1,2-diacylglycerol and inositol 1,4,5-trisphosphate in the 7-thia-8-oxoguanosine-treated B lymphocyte cultures revealed an enhanced formation of these second messengers leading to the activation and possible de novo synthesis of protein kinase C. The relevance of these results are discussed in regard to the transduction mechanism linking these guanosine analog generated signals with subsequent B lymphocyte activation events.