Hirohisa Tsuda

Chromosomal aberrations and morphological transformation in hamster embryonic cells treated with potassium dichromate in vitro.

The addition of K2Cr2O7, at concentrations ranging from 0.1 to 0.5 microng/ml, to hamster total embryonic cells for 24 h, resulted in consistent and drastic chromosomal aberrations including gaps, breaks and exchanges. The above effect, however, was reduced successfully by the addition of a reducing agent, Na2SO3. Among other chromium compounds examined, divalent and trivalent chromium salts were ineffective on chromosome morphology even at a concentration of 3.5 microng/ml as chromium, whereas a hexavalent compound, CrO3, was highly effective. K2Cr2O7 also enhanced the morphological transformation rate in a short-term colony assay, in whicy hamster embryonic cells (1x10(4) cells/60-mm dish) were treated and the morphology was observed 8 to 10 days after the treatment.

Production of Antigen-Specific Human Antibodies from Mice Engineered with Human Heavy and Light Chain YACsa

Our paper describes the introduction of large fragments of both the human heavy and light chain Ig genes into the mouse germline to create a mouse strain capable of producing a broad repertoire of antigen-specific, fully human antibodies. The human immunoglobulin gene sequences were functional in the context of the mouse machinery for antibody recombination and expression, either in the presence or absence of functional endogenous genes. This was demonstrated by their ability to undergo diverse rearrangement, to be expressed at significant levels, and to exclude expression of mouse immunoglobulins irrespective of their copy number or site of integration. The decrease in susceptibility to influence by adjacent genomic sequences may reflect the greater size, variable gene content, or structural integrity of the human Ig YACs and/or the presence of unidentified but important regulatory elements needed for optimal expression of the human immunoglobulin genes and their correct regulation. Our results show that mouse B cells coexpressing human heavy and kappa chains, upon immunization, can produce antigen-specific, fully human antibodies. Furthermore, the human heavy and kappa chain YACs induced differentiation and maturation of the growth-arrested B-cell lineage in mice with inactivated endogenous Ig genes, leading to the production of a diverse repertoire of fully human antibodies at levels approaching those in normal serum. These results suggest the potential value of these mice as a source of fully human antibodies for human therapy. Furthermore, it is expected that such mice would lack immunological tolerance to and thus readily yield antibodies to human proteins, which may constitute an important class of targets for monoclonal antibody therapy. Our findings suggest that the introduction of even larger portions of the human heavy and light chain loci, which should be achievable with the ES cell-yeast spheroplast fusion technology described, will result in strains of mice ultimately capable of recapitulating the full antibody repertoire characteristic of the human humoral response to infection and immunization. The present and future mouse strains may prove to be valuable tools for studying the molecular mechanisms and regulatory sequences influencing the programmed assembly and expression of human antibodies in the normal immune response, as well as the abnormal response characteristic of autoimmune disease and other disorders. The strategy we have described for the introduction of large segments of the human genome into mice in conjunction with the inactivation of the corresponding mouse loci may also have broad applicability to the investigation of other complex or uncharacterized loci.

Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells

With the goal of creating a strain of mice capable of producing human antibodies, we are cloning and reconstructing the human immunoglobulin germline repertoire in yeast artificial chromosomes (YACs). We describe the identification of YACs containing variable and constant region sequences from the human heavy chain (IgH) and kappa light chain (IgK) loci and the characterization of their integrity in yeast and in mouse embryonic stem (ES) cells. The IgH locus-derived YAC contains five variable (VH) genes, the major diversity (D) gene cluster, the joining (JH) genes, the intronic enhancer (EH), and the constant region genes, mu (C mu) and delta (C delta). Two IgK locus-derived YACs each contain three variable (V kappa) genes, the joining (J kappa) region, the intronic enhancer (E kappa), the constant gene (C kappa), and the kappa deleting element (kde). The IgH YAC was unstable in yeast, generating a variety of deletion derivatives, whereas both IgK YACs were stable. YACs encoding heavy chain and kappa light chain, retrofitted with the mammalian selectable marker, hypoxanthine phosphoribosyltransferase (HPRT), were each introduced into HPRT-deficient mouse ES cells. Analysis of YAC integrity in ES cell lines revealed that the majority of DNA inserts were integrated in substantially intact form.

Invitro transformation of newborn hamster cells by sodium nitrite

Summary Addition of sodium nitrite to cell cultures obtained from newborn Syrian hamsters resulted in transformation. The shortest time required for morphological transformation after this treatment was 21 days. Two of 5 transformed cultures produced progressively growing tumors when injected into animals. These tumors were diagnosed as fibrosarcomas. In two control cultures of the same experimental groups, transformed cells appeared 10 weeks or more later than in treated cultures.

High rate of endoreduplications and chromosomal aberrations in hamster cells treated with sodium nitrite in vitro.

As shown by results in this paper, NaNO2 induced endoreduplications and chromosomal aberrations as well as malignant transformation, in hamster cells in vitro, although the carcinogenic action of NaNO2 has not been reported in animals. The mode of action of NaNO2 in mammalian cells requires further investigation.

Effects of diesel exhaust particles on chromosome aberration, sister chromatid exchange and morphological transformation in cultured mammalian cells

Abstract The ability of diesel exhaust particles (diesel tar) from light-duty (LD) and heavy-duty (HD) engines to induce chromosome aberrations, sister chromatid exchanges (SCEs) and morphological transformations is examined. Chinese hamster V79 cells were treated with LD and HD diesel tar for 3 h in order to analyze chromosome aberrations and SCEs. 3T3 cells were used for the morphological transformation test. LD tar induced significant numbers of chromosome aberrations, whereas HD did not. Both LD and HD samples increased the number of SCEs in a dose-dependent fashion, with LD being more potent. In the transformation test, LD tar also induced a significant number of Type III foci, while HD was only weakly active. The transformed cells isolated from these Type III foci produced tumors when injected into nude mice. These results show that LD possesses clear clastogenic and transforming capabilities but that HD is weaker in this regard.

Effect of tumor promoter TPA on spontaneous and mitomycin C induced mitotic recombination in Drosophila melanogaster

It has been proposed that mitotic recombination is involved in tumor promotion. To test this idea, we investigated the effect of a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), on spontaneous and mitomycin C (MMC)-induced mitotic recombination in Drosophila melanogaster. The test system used was the wing-spot assay. Third instar larvae (mwh+/+flr) were treated with MMC (0-0.3 mM) for 6 h and fed media containing TPA (0-10 micrograms/ml), and the wings of surviving adults were analyzed for the presence of mutant spots. The results are: (1) MMC induced twin spots as well as single spots dose dependently (0.03-0.3 mM). (2) TPA itself did not induce twin spots or single spots in the range of doses examined (0.1-10 micrograms/ml). (3) TPA did not enhance the frequencies of twin or single spots induced by MMC. These results indicate that TPA induced neither mitotic recombination nor mutations under these experimental conditions. Thus the results would not support the mitotic recombination theory in tumor promotion. Rather, in this study, TPA posttreatment resulted in reduced frequencies of mutant single spots induced by MMC.

Role of DT diaphorase the cytotoxicity of menadione and 4-nitroquinoline-1-oxide in cultured mammalian fibroblastic cells

The role of DT diaphorase on the cytotoxicity (reduction in colony formation frequency) of menadione and 4-nitroquinoline-1-oxide (4NQO) was examined in two fibroblastic cell lines (Chinese hamster V79H3 cells and NG2 Syrian hamster cells). The addition of dicoumarol (10(-4) M-3 x 10(-4) M), a specific inhibitor of DT diaphorase, resulted in an intensification of the cytotoxicity of menadione, supporting the hypothesis that DT diaphorase protects cells against the oxidative stress induced by quinones. On the other hand, the toxicity of 4NQO was greatly reduced by the addition of dicoumarol (10(-5) M-3 x 10(-4) M), showing that DT diaphorase is the key (or the sole) enzyme involved in the activation of 4NQO in the above cells.

Marked increases in DT diaphorase activity in malignantly transformed Syrian hamster fibroblastic cells

NAD(P)H quinone oxidoreductase (DT diaphorase; EC 1.6.99.2) activity in cultured Syrian hamster fibroblastic cells was measured. The cells examined were classified into three categories: (1) four primary embryonic fibroblasts, (2) three non-malignant immortal cells and (3) four malignantly transformed cells. The results showed that cytosolic DT diaphorase activity in malignant cells was markedly higher than the corresponding activity in non-malignant cells (enzyme activity: (3) much greater than (1) greater than or equal to (2)). The enzyme activity was not influenced very much by the particular phase of cell growth. Thus, the increase in DT diaphorase activity seems to be a good marker for malignant transformation, at least in Syrian hamster fibroblastic cells.

Effects of diesel exhaust particles on chromosome aberration, sister-chromatid exchange and morphological transformation in cultured mammalian cells

The ability of diesel exhaust particles (diesel tar) from light-duty (LD) and heavy-duty (HD) engines to induce chromosome aberrations, sister chromatid exchanges (SCEs) and morphological transformations is examined. Chinese hamster V79 cells were treated with LD and HD diesel tar for 3 h in order to analyze chromosome aberrations and SCEs. BALB/c 3T3 cells were used for the morphological transformation test. LD tar induced significant numbers of chromosome aberrations, whereas HD did not. Both LD and HD samples increased the number of SCEs in a dose-dependent fashion, with LD being more potent. In the transformation test, LD tar also induced a significant number of Type III foci, while HD was only weakly active. The transformed cells isolated from these Type III foci produced tumors when injected into nude mice. These results show that LD possesses clear clastogenic and transforming capabilities but that HD is weaker in this regard.