Advantages of the scanning ion microscopy for mapping halogen corticoids in normal and transformed cells in culture.

The intra-cellular distribution of eight halogen glucocorticoids was investigated by ion microscopy in two cellular varieties of cultured non-cancer cells (fibroblast 3T3) and cancer cells (human breast tumor cells MCF-7). Two types of ion microscopy helped to determine this distribution, a direct imaging ion microscope (SMI 300) with low spatial resolution, and a scanning ion microscope (IMS4F), featuring high resolution, serving to obtain maps representing the intra-cellular distribution of the fluorine elements and drugs present in these monolayer cultured cells. The fluorine images representative of the drugs containing fluorine showed that these drugs are essentially concentrated in the cell nuclei. In these nuclei, the distribution of these drugs is different from that of heterochromatin and of the nucleolus.

Translation of the hepatitis B virus P gene by ribosomal scanning as an alternative to internal initiation.

The hepatitis B virus (HBV) P gene which encodes the reverse transcriptase and other proteins required for replication is expressed on the bicistronic mRNA pregenome which also encodes the capsid protein in its first cistron. Recent results have suggested that the hepadnaviral P gene is translated by internal entry of ribosomes upstream from the P gene, in the overlapping C gene. Using a reporter gene fused to the HBV C or P gene, we demonstrate that the C sequence does not allow internal initiation of translation. Alternatively, our results support a model in which the HBV P gene is translated by ribosomes which scan from the capped extremity of the bicistronic mRNA pregenome. The mechanism by which the ribosomes scan past four AUGs before they initiate translation at the P AUG was analyzed. Our results show that these AUGs are skipped via two mechanisms: leaky scanning on AUGs in a weak or suboptimal initiation context and translation of an out-of-C-frame minicistron followed by reinitiation at P AUG. The minicistron translation allows ribosomes to bypass an AUG in a favorable context that would otherwise be used as a start codon for translation of a truncated capsid protein. Our results suggest that this elaborated scanning mechanism permits the coordinate expression of the HBV C and P genes on the viral bicistronic mRNA pregenome.

MHC class I antigen expression and alterations induced by rIFN gamma in tumor hybrid cell immunogenicity.

Hybridization of a poorly immunogenic tumor cell with an allogeneic cell was performed in order to improve tumor immune response; several variants derived from one hybrid tumor cell were studied. We compared the immunogenicity of these variants and their allogeneic and syngeneic class I antigen expression before and after IFN gamma treatment. Allogeneic class I antigens were weakly expressed in all variants; IFN treatment enhanced their expression similarly in both immunogenic and nonimmunogenic variants. Syngeneic class I antigen expression differed among variants: IFN treatment induced changes in their expression which corresponded to a posttranscriptional event and which could, at least partly, explain the modifications observed in their immunogenicity.

Intracellular localization of drugs in cultured tumor cells by ion microscopy and image processing.

Several drugs, containing a halogen atom, F or Br, that are being used in antiviral or anticancer therapy, were studied for their localization in cultured cells by ion microanalysis. The association allows to reduce the exposure time to define the intracellular localization of the studied element. The topography of the cells is given by the image of the polyatomic ion 26CN-. The image of the distribution of 81Br- or 19F-, coded in another color scale, can be superimposed, giving a polychromic image of the cell, thus showing the intracellular localization of the compound. MCF-7 tumor cells were cultured in the presence of pyrimidine derivatives. 5-Bromo-2'-deoxyuridine (BUdR) and 5-trifluorothymidine (F3TdR) were localized in the nucleus, 5-fluoro-2'-deoxyuridine (FUdR) in the nucleus and only in some nucleoli. The method is simple and rapid, as compared with techniques using radiolabeled compounds, or with immunocytochemical techniques. It is possible to observe two different compounds in the same cell. It could be applied to other compounds containing a halogen atom.

Role of T suppressor cells in the cycling of the immune response against a murine fibrosarcoma.

Antitumor immunity against a fibrosarcoma in C57BL/6 mice was obtained by means of a semi-allogenic somatic hybrid cell derived from the fusion of this C57BL/6 fibrosarcoma (MCB6-1) and A9 cells of C3H origin. In a Winn assay, this immunity could be transferred by T lymphocytes to normal C57BL/6 recipient mice during an early and a late phase after immunization. There appeared to be a transient non-responsive period during which no immunity could be transferred. Injection of cyclophosphamide (CY) into mice before immunization increased the level of immunity during this period, and reconstitution of animals with normal spleen cells abolished the effect of CY. During the non-responsive period, suppressor cells were demonstrated in the spleen: the i.v. transfer of these suppressor cells to normal mice significantly inhibited the induction of antitumor immunity; the suppressive effect was transferred by T lymphocytes of the Lyt-2+ phenotype. No suppressive effect on antitumor protection was observed when suppressor cells were transferred simultaneously with immune T lymphocytes in the Winn assay. From these findings, it appears that T-suppressor cells regulate the antitumor response, interfering with the afferent (induction) arm of the immune response.

Effect of selenium in combination with cis-diamminedichloroplatinum(II) in the treatment of murine fibrosarcoma.

cis-Diamminedichloroplatinum(II) (cis-DDP) is a well-known anticancer agent the use of which is limited by its toxicity. Since it has been demonstrated that selenium is able to combine with metals like cadmium and mercury and to reduce their toxicity, we decided to investigate whether it could reduce the toxicity of platinum. We treated fibrosarcoma-bearing mice with a combination of cis-DDP and selenium. The dose of 2 or 4 micrograms selenium/g animal weight had no effect on tumor growth. The i.p. injection of 16 micrograms cis-DDP/g led to early death of animals. The i.p. treatment of tumor-bearing animals with 2 or 4 micrograms of selenium reduced the early mortality induced by cis-DDP at a dose of 16 micrograms/g. Therefore, the addition of selenium allowed the administration of high doses of cis-DDP, which resulted in an improved antitumor effect. Clonogenic assays following drug exposure showed that selenium had no direct effect on tumor cells and did not modify the antitumor activity of cis-DDP. Electron microscopy showed reduced changes in renal cells when selenium was added to the cis-DDP treatment. Microanalysis showed no accumulation of either selenium or platinum within renal cells. These results suggest that the addition of selenium decreases the nephrotoxicity of cis-DDP.