Quantitative cytofluorimetric determination of cell membrane-associated large tumor antigen on SV40-transformed cells.

The aim of this study was to quantitate the number of cell membrane-located SV40 large tumor antigen (large T) molecules of SV40-transformed cell lines by cytofluorimetric analysis. Five different SV40-transformed cell lines were labelled by either a biotin- or a fluorescein-conjugated monoclonal antibody, PAb1605, which is specific for the large T carboxyterminus. The conjugated-antibody fluorescence signals of the stained large T molecules of transformed cells were measured via cytofluorimetry. Comparison of the fluorescence signals of calibrated beads bearing a known number of fluorescein molecules to the signals of conjugated PAb1605 antibodies bound on microbeads to a defined number of IgG binding sites made it possible to determine the number of antibody-accessible large T molecules per SV40-transformed cell. The numbers (x10(-4)) found per cell were 1.0 (ELONA, hamster), 3.0 (VLM, mouse), 3.5 (mKSA, mouse), 11 (C57SV, mouse), and 5.5 (SV80, human), respectively. Thus, the technique described allows a precise quantitation of surface-exposed, antibody-accessible viral antigen expression.

Monospecific polyclonal anti-anti-idiotypic antibodies to the carboxyterminal undecapeptide of the SV40 large tumour antigen.

The murine monoclonal antibody PAb1605 defines an epitope, peptide Lys(698)-Thr(708) (KT), on the carboxyterminus of the tumour(T)antigen of SV40-transformed cells. In vivo and in vitro experiments had shown that this sequence represents an epitope for both humoral and cellular immune responses. When injected into rabbits PAb1605 induces anti-idiotypic antibodies (Ab-2). Ab-2 beta (internal image type) was purified by adsorption chromatography and characterized by the ability of KT to compete with the binding of ab-2 with ab-1. Murine anti-anti-idiotypic antibodies (ab-3) were obtained by immunization of mice with ab-2 beta. Both ab-1 and ab-3 JgG showed affinities to immunoprecipitated SV40 T antigen by immunoblot analysis and to nuclear SV40 T antigen by the immunofluorescence assay. The binding of ab-3 to SV40 T antigen was completely inhibited by competition with KT. We conclude that the polyclonal ab-3 is of the ab-3 subtype and specific for only one epitope which is represented by KT and defined by ab-1. The results demonstrate that the specificity for a defined peptide epitope of an antibody was conserved even after two consecutive steps of anti-idiotypic-antibody formation in two host species. Since this postulate of network theory could be verified for a sequence of a tumour-associated antigen which represents a B- and T cell epitope, this model is of great interest for further tumour immunological studies.

The cell-binding carboxyterminal undecapeptide of SV40 tumour antigen provides protective cell-dependent immunity.

This paper describes the use of the synthetic carboxyterminal undecapeptide of large SV40 tumour antigen, lys698-thr708 (KT) to protect Balb/c mice against growth of subcutaneously transplanted tumorigenic SV40-transformed cells (VLM). The vaccine was prepared by conjugation of KT with 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide (SPDP). Addition of the SPDP-derivative of KT to syngeneic spleen cells rendered KT covalently linked to free thiol-groups of the cell membranes by the formation of -S-S-CH2-CH2-CO-epsilon-NH-lys698 bonds. Vaccination with KT-conjugated cells was intraperitoneal. Alternatively, KT-conjugated cells were generated in the peritoneum by injection of PDP-KT ((2-pyridyldithio)propionic acid-KT). As a control 60Co-irradiated VLM cells were used. In five experiments all VLM-vaccinated and the majority of the PDP-KT-(or KT-spleen cell)-vaccinated mice were protected against tumour growth. However, mice pretreated with saline, unconjugated spleen cells, free KT, KT conjugated to bovine serum albumin, or KT with incomplete Freund's adjuvant developed tumours. Treatment of PDP-KT-vaccinated mice with anti-CD4 or anti-CD8 immunoglobulin abolished tumour immunity completely. Thus, covalent binding of the carboxyterminal undecapeptide of SV40 tumour antigen to viable, untransformed cells yielded a vaccine which protects Balb/c mice against SV40 tumours.

Nuclear accumulation of p53 in response to treatment with DNA-damaging agents.

A number of agents which damage DNA also trigger the nuclear accumulation of the tumor suppressor protein p53. Here we show the correlation with different p53 detection methods. As an example we investigated the effects of the cancer therapy drug mitomycin C on different mammalian cell lines. Our findings demonstrate that either the immunofluorescence techniques (indirect immunofluorescence staining or flow cytometric analysis) or ELISA or immunoblot assays are useful methods in detecting p53 accumulation. Simultaneously we measured DNA damage with the terminal deoxynucleotidyl transferase assay. Compatible data were obtained. Thus p53 accumulation may be used as indicator of DNA injury.

Covalent immunochemical membrane labeling of viable cells with K698-T708, a simian virus 40 tumor antigen-derived peptide.

The process of covalent immunochemical linking of viable cell membranes with a Simian Virus 40 (SV40) tumor antigen-derived undecapeptide, K(698)PPTPPPEPET(708) (KT), is described. The principle applied was the reaction of the lysine residue, K 698, of the undecapeptide with the succinimidyl moiety of a heterobifunctional linker molecule, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (sulfo-SIAB). Thereby, upon release of N-hydroxy-succinimide, the rest of the linker molecule reacts covalently with the epsilon-NH2 group of lysine. Upon release of pyridyl-2-thion or hydrogen iodide, respectively, the second reactive moiety of the linker is then ready to form a covalent bond with SH-groups of cell membrane compounds. As a result, KT is covalently linked onto the cell membrane by an -SS- or an -S-bond, respectively. Binding is prevented by treatment of the candidate cells with iodoacetamide, an SH-reactive compound. This artificial cell membrane epitope can be demonstrated by surface immunofluorescence and by binding to immunomagnetic beads loaded with PAb1605, a KT-specific monoclonal antibody. Quantitation by cytofluorimetry shows some 10(4) KT molecules bound per cell, a number that is in the range of the number of SV40 tumor antigen molecules of genuine SV40-transformed mammalian cells.

PAb1614, a monoclonal antibody reactive with the tumor antigens of SV40, JC, BK, and polyoma virus, and other JC virus tumor antigen cross-reactive antibodies of the PAb1601-1636 panel.

PAb1614, an SV40-specific monoclonal antibody of the panel PAb1601-1636 reacts with large and small tumor antigens of SV40, BK and JC virus, and with polyoma virus large and middle tumor antigens, but not with the large tumor antigen of the lymphotropic papova virus. Using immunofluorescence and immunoblot competition assays and ELISA with synthetic peptides, it is shown that the epitope is represented by the SV40 tumor antigen undecapeptide, K39-E49. This peptide comprises the tumor antigen consensus sequence, H42-G47, of the polyoma viruses. However, the epitope of PAb1614 probably does not exactly coincide with this hexapeptide. This explains why some cross-reactions are less strong, or absent, as in the case of the lymphotropic papova virus. Further antibodies of the PAb1601-1636 panel that cross-react with the JC virus large tumor antigen are PAb1602, 1604, 1606, 1618, 1621, 1622, 1623, 1624, 1626, 1629, and 1633.

Filtered, tar-free and aerosol-free cigarette-smoke causes accumulation of the tumor-suppressor protein p53 in rodent cells.

Cigarette smoke was filtered with a Cambridge glass fiber filter retaining 99.9% of the tar and aerosol fraction and diluted 1:5 with air. The murine cell line L929 was exposed to this smoke preparation for periods of up to 10 min. Thereafter the following parameters were determined at different times: Nuclear accumulation of the tumor suppressor protein p53 indicating chromatin injury (by immunostaining); apoptotic DNA fragmentation (by DNA end labelling with biotin-16-dUTP in the presence of terminal deoxyribonucleotidyl transferase); the intracellular level of reactive oxygen intermediates (ROI) (by cytofluorimetry with the fluorigenic stain 2',7'-dichlorofluorescin diacetate). After 1 min exposure to 1:5 air-diluted filtered cigarette smoke maximal p53 accumulation occured about 20 h later, whereas maximal DNA fragmentation and apoptosis and maximal ROI levels were found after 10 min of exposure. Obviously, even the diluted, tar- and aerosol-free fraction of cigarette smoke has the potency, after 1 min of exposure only, to exert severe DNA damage, a potential transformation risk for the surviving cell fraction, in murine cell cultures as indicated by stabilization and accumulation of the tumor suppressor protein p53.

Induction of nuclear accumulation of the tumor-suppressor protein p53 by DNA-damaging agents.

Cancer therapy drugs, such as diamminedichloroplatinum (cisplatin), mitomycin C, etoposide and a number of other compounds, as well as energy-rich radiation, are known to act on cellular DNA. These agents are shown to induce nuclear accumulation of the so-called tumor-suppressor protein p53 in fibroblastoid cells, as well as in epithelioid normal and immortalized cells of murine, simian, and human origin. p53 accumulation starts a few hours after treatment and can remain detectable in surviving cells for at least 20 days. Accumulation occurs because of increased p53 protein stability and depends on ongoing translation. It is not the result of enhanced gene expression. A number of cell cycle inhibitors do not affect p53 protein accumulation, suggesting that the process may start from several points in the cell cycle. Since the increase in the nuclear p53 protein levels occurs within a few hours in most of the treated normal diploid cells, it is unlikely that the accumulated p53 protein is derived from a mutated p53 gene. The results obtained are in accordance with the view that the DNA damage-induced p53 accumulation may either inhibit cell growth, allowing DNA repair processes, or, in the case of severe damage, initiate apoptosis.

[Anthrax in Chad: a zoonosis that still exists today]. / Le charbon au Tchad: une zoonose encore d'actualité.

An epidemic of human and animal anthrax raged in Chad mainly in the Department of Chari Baguirmi from September to December 1988, infesting more than 50% of donkeys and horses. 716 human cases have been reported, with 88 deaths. Thanks to a geographical distribution of animal and human prevalence, one sees immediately the interdependency between sanitary state of live-stock and public health. An unusual means of transmission from donkey to donkey by insects as the vector is suggested to explain the intensity of animal epidemics. Two strains of B. anthracis were isolated and described. Systematic annual prophylactic inoculation of the live-stock is recommended, and also resumption of research to create a polyvalent vaccine for cattle plague/peripneumonia and anthrax.