Effects of tamoxifen, droloxifene and 17 beta-estradiol on Rauscher mouse leukemogenesis.

The effects of tamoxifen (TAM), its 3-hydroxy congener droloxifene (DROL) and 17 beta-estradiol were investigated on leukemogenesis induced in BALB/c mice by Rauscher murine leukemia virus (RLV). Multiple applications of each compound, in a dose-dependent manner, resulted in reduced virus titer in the serum, delayed onset of splenomegaly and significant prolongation of survival. Although 17 beta-estradiol proved most effective, prevention of disease was not achieved either by short- or long-term treatment with any of the drugs tested.

A sensitive non-isotopic assay specific for HIV-1 associated reverse transcriptase.

A sensitive non-isotopic assay for specific detection of reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is described using 5-bromo-2'-deoxyuridine triphosphate (BrdUTP) instead of tritiated thymidine triphosphate. After the RT reaction the template primer is degraded by alkaline hydrolysis. Single-stranded poly.(BrdU) is detected in an immunoenzymometric assay using monoclonal anti-BrdU antibodies. The specificity of the assay is demonstrated by the isolation of RT from virus lysate by an insolubilised monoclonal anti-HIV-1 RT antibody prior to the RT reaction. Immunological RT binding leads to a tenfold increase in analytical sensitivity since substances inhibiting the RT reaction can be removed. This non-isotopic assay is some 30 times more sensitive than the classical radioisotopic RT assay. In terms of RT determination, however, there is a good correlation between these tests (r = 0.96). Several filtrations are no longer necessary to remove non-incorporated nucleotides. The test can be adapted to microtitre plates and hence is easy to automate.

[The influence of macrophages on the leukemogenic activity of Rauscher murine leukemia virus]. / Untersuchungen über den Einfluss der Makrophagen auf die leukämogene Wirkung des Virus der murinen Rauscher-Leukämie (RLV).

A single application of trypan blue 3 to 24 hours before or simultaneous with inoculation of the Rauscher leukemia virus (RLV) resulted in enhancement of leukemogenesis with an especially high viremia. Repeated administration of trypan blue after RLV infection resulted in reduced spleen weight. Viremia, however, was also increased compared with controls (no application of trypan blue), but in a lower rate than in mice treated with the virus plus trypan blue. Since trypan blue is known as an inhibitor of the functions of macrophages the results point at an important role of this cell type in preventing infection with this oncogenic retrovirus. In this regard there are differences in the role of macrophages in human immunodeficiency virus (HIV) infections.

[Fine structure studies and identification of lectin receptors on the viral envelope of two HIV-isolates]. / Feinstrukturuntersuchungen und Identifizierung von Lektinrezeptoren auf der Virushülle zweier HIV-Isolate.

The electron microscopic particle findings were compared with the levels of revertase in corresponding samples over a longer period of time, and a good correlation was found. Comparative investigations of the fine-structure of two HIV isolates did not reveal any morphological differences. It can be assumed, on the basis of the comparative studies on lectin receptors using Helix pomatia lectin, that the viral envelopes of the two isolates are equipped similarly with N-acetyl-d-galactosamine. The differences are not significant with mature particles.

[The use of 60Co-gamma ray-sterilized calf serum in cell culture]. / Untersuchungen zum Einsatz 60Co-gamma-Strahlen-sterilisierter Kälberseren in der Zellzucht.

The use of 60Co-gamma-radiation-sterilised calf sera in cell culturing is reported in this paper. Evidence was produced to the effect that 60Co-gamma-irradiation, using a dosage of 3 kGy and a dose rate of 8 kGy/h, of fetal calf serum, neonatal calf serum, and calf serum did not substantially alter the growth-stimulating properties of those sera during 42-day tests. With almost all cell lines and sera used, for all practical purposes, they were identical with the properties of control sera. The following cell lines were used in the experimental programme: one human mammary tumor, MaTu, one human embryonic cell line--E VI, one bat lung cell line--Tb1-Lu, and one human rhabdomyosarcoma--A 204. Growth stimulation was twelve percent below the control value only with Tb1-Lu on Eagle-MEM culturing medium with 3-kGy-irradiation of neonatal calf serum. On the other hand, cell growth was stimulated by 28 percent in A 204 on RPMI 1640 culturing medium, again with 3-kGy-irradiation of neonatal calf serum. Loss of activity by up to 30 percent, depending on the serum used, must be expected from irradiation doses of 10 kGy and 20 kGy which are capable of causing drastic reduction or even complete elimination of serum-borne microorganisms (Bender et al., 1989). Sera irradiated that way would be only conditionally applicable, when it comes to highly vulnerable cell strains.

Further characterization of the leukemogenic activity of haloperidol in mice.

Haloperidol, a butyrophenon, is widely used for the treatment of psychotic disorders in man. Recently we reported that this drug causes, with high incidence, the development of monocytic-myeloid leukemias in male NMRI mice upon 5 X 5 mg/kg i.p. administration. Here we present evidence for the leukemogenic effect of haloperidol in two other strains of mice (XVII AKF1 hybrids, and the low leukemic BALB/c/BOM). The strain-dependent incidence of leukemias ranged both in males and females between 34% (AKR) and 69% (XVII AKF1) with average latencies between approximately 200 (AKR) and 600 (BALB/c) days. On the basis of cytological and cytochemical criteria the predominating type of leukemias was classified as monocytic-myeloid. These leukemic were serially transplantable. Cell-free extracts of leukemic tissues did not induce the disease indicating that no virus was activated by haloperidol. However, when the drug was administered to AKR mice after a suboptimal dose of nitrosomethylurea (NMU), a higher incidence of mixed-type leukemias was observed as with haloperidol alone. NMU alone induced lymphatic leukemias with proven viral involvement. The tumor promoter 12-0-tetradecanoylphorbol-13-acetate did not influence haloperidol-induced leukemogenesis.

Effect of pentosan polysulfate (SP 54) on the reverse transcriptase activity of several retroviruses.

Pentosan polysulfate (SP 54), a low molecular weight sulfated polysaccharide, was studied in vitro for its effect on the reverse transcriptase activity of seven retroviruses. Six of them possess an enzyme with high sensitivity against SP 54, while the enzyme of one virus (bovine leucosis virus) proved to be insensitive within the concentration range tested. In comparison with other polyanionic compounds so far tested, SP 54 seems to be one of the most active in vitro inhibitors of retrovirus-specific reverse transcriptase.

[The effect of phenolic polymers on retroviruses]. / Zur Wirkung von Phenolkörperpolymerisaten auf Retroviren.

Phenolic polymers synthesized by enzymatic oxidation of coffeic acid, chlorogenic acid, and gentisinic acid were found to strongly inhibit RNA-dependent DNA polymerase (revertase) of retroviruses. Except of two type C retroviruses inhibition became reversible by the addition of bovine serum albumin to the exogenous revertase test. The phenolic polymers tested did not influence the propagation of retroviruses in the cell culture. The replication of Rauscher leukemia virus in mice was diminished by a short-time preincubation of virus suspension with coffeic acid polymer (KOP). In contrast, the preincubation of a virus-containing serum with KOP increased the leukemogenic effect of the virus. KOP given to mice at a high dose subsequently to virus inoculation resulted in high revertase activities and in an elevation of spleen weights too.

[The biological effects of coordination compounds of transitional metals. 6. Effect of 4-methyl-2-aminopyridine-palladium chloride and cis-dichlorodiammine-platinum(II) on retroviruses and the virus-associated RNA polymerase of the influenza virus]. / Uber biologische Wirkungen von Koordinationsverbindungen der Ubergangsmetalle. 6. Wirkung von 4-Methyl-2-aminopyridin-palladiumchlorid und cis-dichloro-diammin-Platin(II) auf Retroviren und die virus-assoziierte RNS-Polymerase von Influenzavirus.

The effect on retroviruses of two transition metal complexes of known antiviral activity, 4-methyl-2-amino-pyridine-palladium-chloride (MAP) and cis-dichloro-diammine-platinum(II) (cis-DDP) has been investigated. The experiments included the evaluation of the action of compounds on virus particle-associated reverse transcriptase in exogenous assays, on virus propagation in persistently infected cell cultures and on virus infectivity in mice. In disrupted viruses and in the absence of excess protein, the reverse transcriptase was inhibited by MAP but not by cis-DDP. The same results were obtained when examining the activity of the virus-associated RNA polymerase of influenza virus A/WSN. Both compounds did not inhibit the replication of retroviruses in cell cultures, except at high dose levels which exerted toxic action on both cells and virus formation. The leukemogenicity of Rauscher murine leukemia virus was strongly inhibited when the virus had been incubated with MAP before inoculation. A similar treatment with cis-DDP did not influence viral leukemogenicity. Despite somewhat different results with both compounds tested, we conclude from the present results that the above mentioned compounds cannot be considered as antiretroviral drugs.

[Serum sialic acid levels in cancer, pregnancy and upper respiratory infections]. / Der Sialinsäuregehalt des Serums bei Krebs, Schwangerschaft sowie Infektionen der oberen Atemwege.

A significant increase in sialic acid content is demonstrable in the sera of cancer patients. Our investigations were performed to find out whether the serum sialic acid level is altered also during pregnancy or with infections of the upper respiratory tract. Our results have revealed a slight increase in sialic acid level to occur only in the last trimester of pregnancy. However, this distinct, though statistically insignificant, increase will not lead to misjudgement in regard to cancer diagnosis, or in case control of this disease. By contrast, in 7 out of 15 patients with infected upper respiratory tract the sialic acid content was clearly above the tolerance limit so that false positive findings may possibly be obtained.

Teleocidin, a tumour promoter, stimulates synthesis of primate retroviruses in persistently infected human cells.

The naturally occurring tumour promoter teleocidin produces a pronounced but transient enhancement of the synthesis of extracellular viral particles in human cells chronically infected with simian retroviruses of type C (baboon endogenous virus, simian sarcoma virus) or type D (Mason-Pfizer monkey virus) or a human cell line-derived type D isolate (PMF virus), respectively. The retrovirus-stimulating activity of teleocidin is very similar to that previously described for the tumour-promoting phorbol ester TPA in the same cell systems.

A simplified quick method for determination of sialic acid in serum.

A modification of the method reported by R. J. Shamberger (J. Clin. Chem. Clin. Biochem. 22, 647 (1984) for detection of serum sialic acid is described. The whole procedure takes less than 1 h. It is based on the release of bound sialic acid by heating with 5% perchloric acid. After cooling and brief centrifugation, the supernatant is heated for 15 min with Ehrlich's reagent at 100 degrees C. Thereafter the absorbance of the color developed in the sample is measured at 525 nm.

Suppression of human lymphocyte mitogen response by retroviruses of type D. I. Action of highly purified intact and disrupted virus.

The type D retrovirus PMFV, derived from a human cell line, suppresses the in vitro response of human lymphocytes to different T-cell mitogens as well as the mixed lymphocyte reaction. The suppressive effect is virus-specific and the active fraction copurifies with the virus particles. The suppression is produced by both crude and highly purified intact and disrupted virus preparations. However, the suppressive activity of virus disrupted by ether or a detergent is higher as compared with intact virus. The absence of any factors cytotoxic for lymphocytes or lymphoblasts in the suppressive virus preparation is shown by different methods.

Suppression of human lymphocyte mitogen response by retroviruses of type D. II. Non-activity of Mason-Pfizer monkey virus versus activity of human cell line derived virus PMFV.

In contrast to the human cell line derived type D retrovirus PMFV, the Mason-Pfizer monkey virus (MPMV) does not suppress the mitogen response of normal human lymphocytes. Both viruses have been propagated on the same cell lines and purified by the same methods. MPMV did not contain a factor able to abolish PMFV-induced suppression of the mitogen response. Neither could MPMV suppress the mitogen response of lymphocytes from rhesus monkeys or baboons. PMFV however inhibited their reactivity.

Enhancement of primate retrovirus synthesis by tumour promoters.

The tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and teleocidin were found to be effective in stimulating the synthesis of primate retroviruses in chronically infected human cells. Production of baboon endogenous virus, simian sarcoma virus of woolly monkeys, Mason-Pfizer virus of rhesus monkeys and of a type D isolate from a human cell line was evaluated by assaying particle-associated reverse transcriptase activities in culture fluids of cells grown in the presence or absence of tumour promoters. Both TPA and teleocidin caused a significant but transient increase in virus production, as well as cytomorphological changes in the following infected cell types: human embryonic kidney, Tu 197 human ovarian carcinoma cells, NC 37 human lymphoblastoid line. However, infected A 204 human rhabdomyosarcoma cells were not modified by these promoters. The stimulation of virus production reached its maximum after two to four days, at which time virus production was three to forty times higher than that in controls. The optimal concentration of tumour promoters was 5-10 ng/mL. 12-O-Retinoylphorbol-13-acetate produced a similar but somewhat weaker effect. Control experiments demonstrated that enhancement was specific to those viruses that chronically infected each cell type.

Biochemical studies of primate retroviruses.

In the present paper, recent biochemical studies of retroviruses carried out in our laboratory are summarized. Protein compositions, peptide maps of internal structural proteins, neighborhoods of major structural proteins, and serological properties of reverse transcriptases of type D virus isolates from human cells (including Graffi's isolate termed PMFV and also isolates from HeLa- and HEp-2 cells) were compared with those of type D viruses from Old World (Mason-Pfizer virus of rhesus monkeys, langur virus) and New World (squirrel monkey retrovirus) monkeys. The results provide various new informations on, and further demonstrate the diversity of primate type D viruses. Other studies showed that tumor promoting agents (phorbol ester TPA, indole alkaloid teleocidin) are able to considerably increase, in a transient manner, the production of type C and type D primate retroviruses in persistently infected human cells. From experiments demonstrating a disintegrating activity of chelating agents (EDTA, EGTA) and certain psychoactive drugs (including trifluoperazine) on various primate and nonprimate retroviruses it is concluded that divalent cations, probably Ca2+ ions, and possibly also cation-binding proteins are associated with retroviral membranes and that complexing of these components results in loss of viral infectivity.

A simple method for purification of retroviruses using polyacrylnitrile.

A simple method has been developed that allows the concentration and partial purification of retroviruses from both small and large volumes of culture fluid in a comparatively short period of time. The method is based on the property of polyacrylnitrile (PAN) to adsorb preferentially nonviral proteins. Retroviruses purified by this procedure exhibit rather high specific reverse transcriptase activities and a good appearance in electron micrographs and protein patterns.

Leukaemogenic and mutagenic activity of the butyrophenon, 4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidinyl]-1-(4-fluorophenyl)-1-butanone (haloperidol).

Development of mainly monocytic-myeloid leukaemias was observed in two strains of mice upon i.p. administration of 4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidinyl]-1-(4-fluorophenyl)-1-butanone (haloperidol), an antipsychotic drug. Haloperidol was also shown to be mutagenic in the Ames Salmonella test system.