An ELISA system for evaluating antiretroviral activity against Rauscher murine leukemia virus.

A system for evaluating the activity of antiviral agents against Rauscher murine leukemia virus (R-MuLV) has been developed using an enzyme linked immunosorbent assay technique. The activity of various antiviral compounds demonstrated in this assay system has been compared to their activity in the UV-XC plaque reduction assay, which has been used historically for evaluating anti-R-MuLV compounds. The assay is based upon detection of R-MuLV encoded p30 protein production in virus infected murine cells. The assay reagents are readily available and the assay system is amenable to automated data collection systems. Cytotoxicity evaluations are conducted in parallel to the Rauscher MuLV ELISA assay in order to assess drug-induced reductions in cell viability. Cytotoxicity evaluations are important to interpretation of the ELISA results since reductions in cell viability reduce viral protein production which would indicate an antiviral drug effect. This system is less sensitive than the classical UV-XC plaque reduction assay; however, it does offer an alternative to the time-consuming and labor-intensive plaque assay.

Antileukemic activity of Viva-Natural, a dietary seaweed extract, on Rauscher murine leukemia in comparison with anti-HIV agents, azidothymidine, dextran sulfate and pentosan polysulfate.

An antileukemic activity of partially purified polysaccharide of an edible seaweed. Viva-Natural, against Rauscher murine retrovirus-induced erythroleukemia has been demonstrated. This antileukemic effect is compared with standard anti-human immunodeficiency virus (HIV) agents, azidothymidine (AZT), dextran sulfate and pentosan polysulfate. Pretreatment with Viva-Natural, as an immunomodulator, on day 3 prior to the virus inoculation demonstrated definite prophylactic activity, while pretreatment with the other three anti-HIV agents showed no prophylactic activity. The replication of Rauscher virus in BALB/3T3 cell cultures accompanied by direct cytopathic effect (syncytia formation) was suppressed in the presence of Viva-Natural or the other anti-HIV agents in the culture medium. In spite of the antiviral potentials of the four agents in vitro, only Viva-Natural and AZT demonstrated therapeutic efficacy against Rauscher leukemia in mice.

Differential inhibition of the activities of reverse transcriptase and various cellular DNA polymerases by a traditional Kampo drug, sho-saiko-to.

A traditional Kampo drug, Sho-saiko-to, composed of several herb extracts, differentially inhibited the activities of reverse transcriptase and human cellular DNA polymerase alpha and beta. Reverse transcriptases from murine leukemia virus and human immunodeficiency virus were inhibited by over 80% and 50%, respectively, in the presence of 100 micrograms/ml Sho-saiko-to, whereas DNA polymerase alpha was much less sensitive to inhibition by this drug than were the reverse transcriptases. DNA polymerase gamma was not inhibited by this drug at concentrations of up to 500 micrograms/ml. Only DNA polymerase beta was moderately inhibited by Sho-saiko-to. Thus, it has been shown that the inhibition by Sho-saiko-to is relatively specific for reverse transcriptase and that the drug contains as yet unidentified inhibitory substance(s) for reverse transcriptase.

Murine models for antiretroviral therapy.

Rauscher murine leukemia virus (RLV) infection of adult mice results in splenomegaly that is proportional to the virus titer. We have used this model to study the therapy of chronic viremia as well as chemoprophylaxis after viral exposure, both with single agents and with combination therapy. We showed that prompt initiation of effective antiviral therapy after viral exposure can prevent de novo infection. We have also developed a murine neurotropic retrovirus model which allows analysis of candidate antiviral agents for activity across the blood-brain barrier. This model can also be used to study therapeutic approaches to retroviral infections acquired at midgestation or during the neonatal period.

Antiretroviral activity in a marine red alga: reverse transcriptase inhibition by an aqueous extract of Schizymenia pacifica.

An aqueous extract from the marine red alga, Schizymenia pacifica has been tested in a cell free system for its effect on reverse transcriptase from avian retrovirus (avian myeloblastosis virus), and mammalian retrovirus (Rauscher murine leukemia virus). The extract inhibited reverse transcriptase from both these retroviruses but showed almost no effect, if any, on the activity of cellular DNA polymerase alpha and RNA polymerase II in vitro. Consequently it is unlikely to have an adverse effect on the growth of cultured cell. The inhibitory activity of the extract was stable over a relatively wide pH range (pH 1-11) and was not lost after pronase digestion. Inhibitory activity of the extract was lost after boiling at 100 degrees C in 0.67 N HCl, and after treatment with 100 mM NaIO4. The active principle in the extract has an apparent molecular weight in excess of 100,000 daltons. This new reverse transcriptase inhibitor is probably a polysaccharide.

[Selenium compounds and carcinogenesis]. / Selenverbindungen und Kanzerogenese.

Starting with a brief outline of the general toxic effects of selenium compounds, their biological importance for the organism as a trace element and with an analysis of the different hypotheses on the action mechanism of selenium and selenium compounds, a survey is provided in which way selenium compounds may influence malignant transformation and related processes in vivo and in vitro. Furthermore, observations are viewed on the effects of selenium compounds on spontaneously developing, chemically or virally induced tumors. Based on the data available so far it cannot, at present, be assessed whether selenium and its compounds can be used in the future for chemoprevention of cancer.

Two-stage carcinogenesis in vitro: transformation of 3-methylcholanthrene-initiated Rauscher murine leukemia virus-infected rat embryo cells by diverse tumor promoters.

A modified in vitro transformation assay demonstrated potential for use in the identification and study of tumor promoters. Rauscher murine leukemia virus-infected F344 rat embryo cells were transformable by various chemical carcinogens when they were administered at appropriate doses. Cells treated with subeffective doses of 3-methylcholanthrene did not transform. However, when these cells were regularly treated with 12-O-tetradecanoylphorbol 13-acetate, sodium phenobarbital, limonene, oleic acid, lauric acid, or saccharin, transformation was observed. Thus several dhemically diverse in vivo tumor promoters behaved as promoters in this in vitro system. This assay appeared to be useful for in vitro efforts to identify promoters and may be of value in studies on the mechanisms of action of these cocarcinogens.

Overview and status of in vitro transformation.

The development of standardized assay procedures has permitted the exploitation of cell culture systems as bioassay tools for the detection of chemical carcinogens. These systems fall generally into 3 classes: diploid cell strains, Syrian hamster embryo cells; cell lines, mouse BALB/c-3T3 and mouse C3H-10T1/2; and cells+virus, Fischer rat cells infected with Rauscher leukemia virus and Syrian hamster embryo cells infected with adenovirus. The results accumulated to date show a good correlation between transformation response in cell culture and carcinogenicity of chemicals in whole animal studies. The major advantages of these systems are their relative brevity (10 days-6 weeks) and resultant low costs, their agreement with whole animal bioassays, and their direct biological relevance to the carcinogenic process. The present major disadvantages are the uncertain nature of the metabolic capabilities of the target cells and the lack of a metabolic activation system that is reliable and adaptable for routine bioassays. The development of epithelial cell systems such as breast, liver, lung, and skin may solve the problem of carcinogen metabolism as well as provide target cells that are representative of major organ sites for cancer in man. The rational use of cell culture bioassays for neoplastic transformation is a valuable component of the toxicological armamentarium to assess risk to humans from exposure to chemicals.

Therapeutic activity of pretazettine on Rauscher leukemia: combination of antiviral activity and cellular protein inhibition.

Pretazettine hydrochloride (PTZ) has been found to inhibit protein synthesis, without being inhibitory to DNA and RNA, in Rauscher leukemic blood cells in mice for at least 6 h after its administration. With comparison to Virazole and cycloheximide, the specific anti-Rauscher virus activity of PTZ has been demonstrated only in acutely-infected NIH/3T3 cells but not in chronically-infected cells. It is not certain that the inhibitory action of PTZ on reverse transcriptase is contributory to its therapeutic activity in leukemic mice.

Therapeutic activity of pretazettine, a narcissus alkaloid on Rauscher leukemia: comparison with tazettine and streptonigrin.

The therapeutic activity of narcissus alkaloid pretazettine HC1 (PTZ) on established Rauscher leukemia has been demonstrated and compared with the isomer tazettine (TZ) and an antibiotic, streptonigrin (SN). PTZ and SN showed remarkable prolongation effect on the life span of the leukemic mice and the antiviral activity has been confirmed in mouse 3T3 cells infected with Rauscher virus. TZ showed no significant activity in the leukemic mice and was inhibitory to the virus growth in the cells at much higher doses than PTZ. It is suggested that the stereochemical rearrangement from PTZ to TZ inactivates the biological activity of PTZ.

Effect of dietary selenium on tumor induction by an oncogenic virus.

Mice exposed to selenium-supplemented or -deficient rations were inoculated with an oncogenic virus, Rauscher leukemia virus (RLV), Splenic lesions were not altered by dietary selenium supplementation or depletion. It is concluded that selenium does not affect neoplasia induced by RLV in mice.

Revistin found by screening for inhibitors of reverse transcriptase of an oncogenic virus.

Revistin, a substance that strongly inhibits the reverse transcriptase activity of murine leukemia virus in our screening system, was obtained from a cultured broth of a soil streptomyces which was closely related to Streptomyces filipinensis. The assay method for the activity was based on the inhibition by a test material of the incorporation of 3H-dTMP into DNA synthesized by the reverse transcriptase of an oncogenic RNA virus. Crude revistin was isolated by serial procedures of salting out with ammonium sulfate and precipitation with cetylpyridinium chloride. The crude material showed neither antibacterial nor antifungal activity. It exhibited against splenomegaly in mice caused by Rauscher leukemia virus infection.

Combination chemotherapy of Rauscher leukemia and ascites tumors by narcissus alkaloid with standard drugs and effect on cellular immunity.

The therapeutic activity of the narcissus residual alkaloid A-2 against Rauscher leukemia has been compared with 10 standard anticancer drugs, and synergistic or additive combination pairs have been selected using a viral leukemia and two transplantable tumor systems. An increased beneficial effect has been demonstrated by a combination of the alkylating and DNA-binding agents and the alkaloid against the three malignant tumors, while a beneficial effect by combining the alkaloid and the antimetabolites (either 6-MP or 5-azacytidine) was seen only against the viral leukemia. The alkaloid has no suppressive activity against cellular immunity as tested by PHA reactivity and allogeneic tumor rejection systems.