An activity that transforms NIH 3T3 cells was generated by the in vitro modification of plasmids containing the human c-Ha-ras-1 proto-oncogene with the synthesized ultimate carcinogen, 2-acetoxyamino -6- methyldipyrido [1, 2-a: 3', 2'-d]-imidazole (N-OAc-Glu-P-1). DNAs isolated from the transformed cells were analyzed by restriction fragment length polymorphism (RFLP) assay using the restriction enzyme Msp I. Of fourteen transformants studied, six contained a mutation in the region of the CCGG sequence of the eleventh and the twelfth codons, in which GG corresponds to the first two nucleotides of the twelfth codon. Transforming activity was also generated by the chemical modification of the plasmids with 4-acetoxyaminoquinoline N-oxide (N-OAc-4AQO). The results clearly indicate that formation of NA adducts with N-OAc-Glu-P-1 or N-OAc-4AQO causes the induction of transformation of mammalian cells.

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Initiating activities of the secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA), were examined in terms of the induction of γ-glutamyltranspeptidase(GGT)-positive foci in rat liver. A rapid induction model of enzyme-altered foci was utilized. GGT-positive foci were clearly induced by DCA ranging from 0.05% to 0.5% in the diet. In contrast, the induction of GGT-positive foci by LCA was less pronounced and was not dose-dependent. These results suggest that DCA possesses initiating activity.

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A new factor capable of inducing differentiation of human leukemic cell line U-937 into macrophages was found and partially purified from the conditioned medium of U-937 previously treated with 12-O-tetradecanoylphorbol 13-acetate. The purification procedure included ultrafiltration, DEAE-Sephacel and butyl-Toyopearl column chromatography. The purified factor gave a major band of protein with a molecular weight of 67, 000 daltons which coincided with the biological activity of differentiation-inducing factor, and it was not adsorbed on a concanavalin A column. These results suggest that this factor is distinct from other differentiation-inducing factors.

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The seed oil of Jatropha curcas L. was shown to contain skin tumor promoters in a two-stage mouse carcinogenesis experiment. By using the irritant test on mouse ear to monitor activity, the “irritant fraction” was partially purified from the methanol extract of the seed oil by column chromatographies on Florisil and Sephadex LH-20. The irritant fraction obtained induced ornithine decarboxylase in mouse skin and inhibited the specific binding of ³H-12-O-tetradecanoylphorbol-13-acetate to a particulate fraction of mouse skin. After initiation with 7, 12-dimethylbenz[a]anthracene (DMBA), this “irritant fraction” induced tumors in the skin of 36% of the mice tested in 30 weeks. Tumor incidences in the groups treated with DMBA alone and “irritant fraction” alone were 7% and 13% in week 30, respectively. Since the skin of Thai people comes into direct contact with this seed oil, an epidemiological study on human skin cancer in Thailand is indicated.

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The multipotenitial carcinogen N, N'-dibutylnitrosamine (DBN) was given to F344 male rats for 2 weeks at three dose levels to study the concentration dependence of its organ specificity. Groups of 20 rats were given water containing 0.25, 0.125 or 0.063% DBN for 2 weeks and then tap water only to drink until they were killed 52 weeks after the start of the experiment. DBN induced preneoplastic lesions in the liver, esophagus, forestomach and urinary bladder. Carcinoma was found only in the liver. Induction of preneoplastic focal hepatocyte lesions positive for the P-form of glutathione S-transferase (GST-P) was quantitatively dependent on the dose of DBN. In the urinary bladder, the incidences of papillary or nodular hyperplasia (PNH) and papilloma of transitional cells tended to increase at higher doses. The incidence and number per unit length of basal cell-type hyperplasias of the esophageal epithelium were significantly higher in all DBN groups than in the control group, though the increase did not show a clear dose-dependency. The incidence of epithelial basal cell hyperplasia of the forestomach was significantly increased at all doses and the increase was apparently dose-related. These results indicate that even in a short-term experiment, DBN exerted multipotential carcinogenic effects. Thus, this system could be used for assay of the modifying effects of compounds administered subsequently on carcinogenesis in different organs.

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VAK3, VAK4 and VAK5 monoclonal antibodies (mAbs) were raised by using disrupted purified human immunodeficiency virus (HIV) as an antigen. These mAbs exclusively reacted with an HIV-infected cell line, H9/HIV, and did not react with human T cell lymphotropic virus (HTLV)-I or -II infected cell lines. Strip radioimmunoassay based on the Western blotting technique revealed that these mAbs recognize a single band that corresponds to a 24-kd (p24) core protein of HIV when disrupted viruses were used as antigens. When cell lysates of H9/HIV were used as antigens, these mAbs gave two bands with molecular weights of 40kd (p40) and 57kd (p57) in addition to p24. VAK5-conjugated Sepharose 4B beads precipitated non-glycosylated proteins, p24, p40 and p57 from [L-³⁵S] methionine-labeled H9/HIV. P57 and p40 appear to be a precursor and an intermediate precursor of p24, respectively. Purified IgG of VAK3 and VAK4, and the serum from an individual infected with HIV inhibited the binding of VAK5 mAb to HIV. These findings suggest that p24 of HIV contains structure(s) with strong antigenicity.

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Non-simultaneous primary malignant tumors in five different organs were detected in a case of cancer family syndrome, and the patient's offspring were found to have colorectal cancers during follow-up. The first cancer in the patient was found in the transverse colon at the age of 47, and partial colectomy was carried out. The second and third were cancers of the cecum and gallbladder which were found concurrently at the age of 61, and ileocecal resection and cholecystectomy were carried out simultaneously. The fourth was cancer of the urinary bladder which was found at the age of 66, and partial cystectomy with interstitial irradiation using a radon seed in the residual urinary bladder was carried out. The fifth was cancer of the stomach at the age of 72, and subtotal gastrectomy was carried out. All of the resected specimens were demonstrated to be different carcinomas histologically. The father and 5 of the 7 siblings of this patient suffered from gastrointestinal or uterine cancers. Furthermore, two daughters of this patient were affected with cancer; the younger with cancer of the descending colon at the age of 47, and the elder with cancers of the transverse colon and rectum at the age of 54. Thus, familial clustering of cancer was shown. Genetic characteristics may have contributed to the development of multiple primary cancers in this family.

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This paper reports the presence of GM2 ganglioside containing N-glycolylneuraminic acid (NeuGc) in human colon cancer tissues. GM2(NeuGc) was detected by two-dimensional thin layer chromatography (2d-TLC)/enzyme-immunostaining using affinity-purified chicken antibody against GM3(NeuGc) and horseradish peroxidase-conjugated rabbit anti-chicken IgG antibody. Like usual GM2 ganglioside containing N-acetylneuraminic acid (NeuAc) isolated from Tay-Sachs brain, GM2(NeuGc) in colon cancer could be converted into GM3(NeuGc) by human kidney β-N-acetylhexosaminidase A in the presence of a GM2-specific activator protein isolated from guinea pig kidney. Three of 7 specimens of Hanganutziu-Deicher (HD) antigen-positive human colon cancer tissues so far examined expressed this unique ganglioside. In order to detect and determine specifically GM2(NeuGc) on human colon cancers, specific antibody against GM2 (NeuGc) has been prepared by immunizing chickens. By a sensitive TLC/immunostaining method using the antibody, the amounts of the antigen were determined to be 0.3-3% of total lipid-bound sialic acid. NeuGc-containing gangliosides were also detected in meconium and fetal intestinal tissues. Three species of antigenic gangliosides in pooled meconium were tentatively identified as GM3(NeuGc), sialylparagloboside and sialylhexaosylceramide on the basis of their migration positions on 2d-TLC and the results of endo-β-galactosidase treatment. GM3(NeuGc) was the sole HD-active ganglioside in fetal intestinal tissue from one of 3 individuals tested; the other two showed no HD-active ganglioside at all. GM2(NeuGc), however, could not be detected in either meconium or fetal tissues so far examined, suggesting that this unique ganglioside is a tumor-specific antigen, at least for human intestinal tissues.

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The activities of T cells and antigen-presenting cells in spleen from tumor-bearing mice were studied in vitro. The in vitro induction of trinitrophenyl (TNP)-specific proliferative T cell responses and TNP-specific cytotoxic T cell responses was markedly impaired in spleen cells from X5563 plasmacytoma-bearing C3H/He mice. The activity of TNP-hapten presentation to proliferative T cells and cytotoxic T cells was also impaired in spleen from tumor-bearing mice. Suppressor macrophage activity was not observed in spleen from tumor-bearing mice, because the addition of TNP-modified spleen cells from tumor-bearing mice to TNP-modified spleen cells from normal mice did not suppress the activity of TNP-hapten-presenting cells from normal mice and the addition of indomethacin, an inhibitor of prostaglandin synthesis, to the culture of T cells with TNP-modified spleen cells from tumor-bearing mice did not restore their activity. The population of I-region associated antigen (Ia)-positive cells in spleen macrophages decreased in tumor-bearing mice. Furthermore, the production of interleukin 1 by spleen macrophages was also impaired in tumor-bearing mice. These results suggest that one of the mechanisms leading to immunological abnormality in the tumor-bearing host is mediated by the dysfunction of Ia-positive antigen-presenting cells.

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The effect of in vivo administration of recombinant human interleukin 1 (IL-1) on T cell functions in tumor-bearing mice was studied using an in vitro assay system. The in vitro induction of trinitrophenyl (TNP)-specific cytotoxic T cell and proliferative T cell responses from spleen cells was impaired in X5563 plasmacytoma-bearing C3H/He mice. However, the administration of IL-1α or IL-1β to tumor-bearing mice restored T cell functions in a dose-dependent manner. Antigen-presenting activities of spleen cells in tumor-bearing mice for T cell activation were not restored by the administration of IL-1. The activities of cytotoxic T cells and cytostatic T cells specific for X5563 cells were also enhanced by the administration of IL-1. Furthermore, in IL-1-treated mice, NK cell activity of spleen cells detected in terms of the killing of Yac-1 cells was also restored. In accordance with these results, the growth of X5563 cells was significantly inhibited and the lymphocytes from IL-1-treated mice specifically inhibited the growth of tumor cells. These results suggest that the in vivo administration of IL-1 restored the impaired T cell and NK cell functions in tumor-bearing mice and activated protective immunity against tumor cells. Thus, recombinant IL-1 can be applied for tumor immunotherapy.

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Proteose peptone-induced peritoneal exudate macrophages (PEM) from C3H/HeJ mice do not respond to lipopolysaccharide (LPS) in vitro in terms of activation for tumor cytotoxicity. This unresponsiveness of PEM was overcome by addition of culture supernatant of normal mouse spleen cells stimulated with insoluble concanavalin A. The supernatant component responsible for the restoration of PEM sensitivity to LPS was indicated to be interferon (IFN)-γ, because pretreatment of the supernatant with anti-mouse IFN-γ antiserum, but not with anti-IFN-(α+β) antiserum, abolished the supernatant activity and because recombinant murine IFN-γ (rIFN-γ) could replace the supernatant effect. Not only the LPS activation of tumor cytotoxicity of PEM but also the sensitivity of PEM to the lethal toxicity of LPS was restored by rIFN-γ. IFN-γ action in restoring the LPS responsiveness of PEM was mimicked by a calcium ionophore, A23187, but not by phorbol 12-myristate 13-acetate. These results suggest that IFN-γ can restore LPS responsiveness of PEM from C3H/HeJ mice and that elevation of the intracellular calcium level is involved in the action of IFN-γ.

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Human alveolar macrophages (AM) obtained by bronchoalveolar lavage from healthy donors were examined for ability to cause lectin-dependent tumor cell killing. Of five plant and two animal lectins tested, only one lectin, wheat germ agglutinin (WGA), induced significant and reproducible lectin-dependent macrophage-mediated cytotoxicity (LDMC) against human bladder cancer (T-24) cells. There was no significant difference between the LDMCs of AM and blood monocytes. All 6 tumor cell lines tested were sensitive to various extents to LDMC induced by WGA. Quantitative analysis with WGA-FITC conjugate showed the presence of various levels of receptors for WGA on the surface of AM, monocytes and tumors. A relatively good correlation was found between the sensitivities of the tumor cells to LDMC mediated by AM and the numbers of receptors for WGA. Pretreatment of AM or monocytes with LPS did not affect their LDMC. These results indicate that a plant lectin, WGA which binds to both human AM and tumor cells, renders human AM cytotoxic to allogeneic tumor cells by a different mechanism(s) from that involved in the nonspecific tumor cytotoxicity of activated macrophages.

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