Peroxisomes were present in trabecular carcinoma and adenocarcinoma induced by 3'-methyl-4-(dimethylamino)azobenzene in the liver of rats. Peroxisomes in well-differentiated trabecular carcinoma (type I) resembled more or less those in hepatocytes in their electron microscopic features, but were considerably small in number. Poorly differentiated trabecular carcinoma (type II) and adenocarcinoma contained peroxisomes in far smaller number than in the well-differentiated trabecular carcinoma, or frequently showed no peroxisomes. Peroxisomes in poorly differentiated trabecular carcinoma and adenocarcinoma were small in size, contained scanty matrix in general, and almost lacked crystalloid nucleoids; however, they were easily identified by electron microscopic cytochemistry of catalase. Catalase activity of these tumors was significantly lower than in the liver tissues. These tumors did not respond to ethyl chlorophenoxyisobutyrate either by proliferation of peroxisomes or by elevation of catalase activity. It is thus suggested that the cellular mechanisms for regulating the formation of peroxisomes and synthesis of the enzyme involved are impaired in the tumor cells.

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The incubation of dimethylnitrosoamine (DMNA) in the presence of rat liver microsomes leads to production of formaldehyde, formic acid, methylamine, and N-methylhydrazine. When pH 5-enzymes are added to the medium there is also the formation of N-methylhydroxylamine and N, N-dimethylhydrazine. The last compound is the only metabolite produced, to a lesser extent, by the pH 5-enzymes.
Thus, the denitrosated or non-denitrosated metabolites are produced either by an oxidative dealkylation and by a reduction of DMNA, catalysed by microsomal and cellular soluble enzymes.

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Decomposition of dimethylnitrosoamine (DMNA) by chemical and physical agents was further investigated. Both photoirradiation with sunlight or ultraviolet ray and reductive reactions under acid conditions (likely occurring in the stomach) led to the formation of formaldehyde, formic acid, and N-methylhydrazine, in addition to denitrosated compounds such as methylamine, dimethylamine, and N-methylhydroxylamine. N-Methylhydrazine was the only compound which was not detected by photoirradiation under neutral conditions.
The agreement between physicochemical and metabolic degradation products and the possible biological meaning are discussed together with the problem of environmental contamination by the nitroso compound.

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Peritoneal cells from C3H/He mice immunized against syngeneic MM46 ascites tumor cells specifically lysed these tumor cells in vitro, while spleen cells or lymph node cells from immunized hosts were completely ineffective. The effector cells differed from lymphocytes in their morphological characteristics, property of adherence, phagocytic activity, and sensitivity to anti-θ serum. The cytolytic action of the peritoneal cells was not due to cytotoxic lymphocytes, but to macrophages. These macrophages lysed tumor cells in co-operation with a factor derived from immunoglobulin-bearing lymphocytes. Neither macrophages from immunized hosts nor the factor from lymphocytes alone had a cytolytic action on the target cells. Normal macrophages showed some cytolytic activity in the presence of the factor from lymphocytes.

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Aryl hydrocarbon hydroxylase activity in Morris hepatoma 5123D is very low, but apparently increases by the administration of 3-methylcholanthrene, as demonstrated in the host liver, small intestine, and lung from the rats bearing this tumor. The peak level of the increased activity in tumor appears 48hr after the initial administration when the repeated treatment is given intragastrically every 24hr. 7, 8-Benzoflavone increases the enzyme activity in tumor and lung, but not in liver and small intestine. Among the subcellular fractions of the tumor cells, induction of the enzyme by 3-methylcholanthrene was observed in microsomes and nuclei, and the increased level of the activity was higher in microsomes than in nuclei.

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Properties of aryl hydrocarbon hydroxylase in the microsomes were compared between Morris hepatoma 5123D and the host liver from rats bearing this tumor. Requirement of NADPH for the assay of the enzyme activity was observed, compared to that of NADH, and also the additive effect of NADH on the requirement of NADPH was found in the tumor and liver. Curve of pH optimum of the enzyme activity in tumor and liver differed between the rats treated with corn oil and those with 3-methylcholanthrene, indicating a slight shift of the peak value to alkaline pH in the latter. The same values of the apparent K_m for NADPH and NADH were shown for the enzyme from the liver and tumor even 24hr after the treatment with 3-methylcholanthrene, but a difference in the apparent K_m for benzo[a]pyrene was demonstrated between the tumor and the host liver, showing 3.6-6.6μM in the former and 9.1-20μM in the latter. By the addition of 7, 8- or 5, 6-benzoflavone to the assay medium for the tumor, the induced enzyme was inhibited noncompetitively, and the constitutive enzyme was enhanced, as demonstrated in the host liver. As observed in the induced enzyme in both tissues, cyclohexene oxide and 1, 1, 1-trichloropropane oxide slightly increased the activity of the constitutive enzyme in the tumor, in contrast to its inhibition in the host liver.

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Changes in cell surface structure by viral transformation were studied by examining changes in the binding of various lectins differing in carbohydrate specificities. Binding of lectins was assayed directly using cells grown on coverslips. The following ¹²⁵I-lectins were used: Concanavalin-A (specific for glucose and mannose), wheat germ agglutinin (specific for N-acetylglucosamine), castor bean agglutinin (specific for galactose), Wistaria floribunda agglutinin (specific for N-acetylgalactosamine), and soybean agglutinin (specific for N-acetylgalactosamine).
Cells of a clone, SS7, transformed by bovine adenovirus type-3, were found to bind 5 to 6 times more Wistaria floribunda agglutinin than the normal counterpart cells (clone C31, from C3H mouse kidney). In contrast, the binding of soybean agglutinin, which has a sugar specificity similar to Wistaria floribunda agglutinin, to normal and transformed cells was similar. The binding of wheat germ agglutinin and castor bean agglutinin, respectively, to normal and transformed cells was also similar. However, normal cells bound twice as much concanavalin-A as transformed cells. Only half as much Wistaria floribunda agglutinin was bound to transformed cells when they had been dispersed with EDTA. These changes in the number of lectin binding sites on transformation are thought to reflect alteration of the cell surface structure.
The amount of lectins bound per cell decreased with increase in cell density, especially in the case of binding of Wistaria floribunda agglutinin to normal cells.

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Inactivation of antitumor antibiotic, Neocarzinostatin, which occurs in vivo or during incubation with serum in a test tube, was found to depend on the dose of serum and incubation period. The inactivation process was accompanied with degradation of Neocarzinostatin to a smaller molecular size with concomitant loss in antigenic activities at a slower rate. Apparent increase, close to about 250% of control, in the antibiotic activity was found before the inactivation, in the presence of diisopropyl fluorophosphate, or about 200% in the presence of N-ethylmaleimide. The present findings suggest that the degradation process partly depends on proteolysis by serine type proteinase(s) and partly on the involvement of sulfhydryl groups. These results imply two possible applications; one for chemotherapy with Neocarzinostatin in combination with inhibitors of proteolysis and the other for drug design through the modification of amino acid residues on Neocarzinostatin molecules which provide the enzyme binding sites such as lysine and arginine for the proteolytic catalysis.

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Electronic interaction of DNA with nine benzacridine derivatives was studied. The interaction system of these benzacridines and DNA was found to show a marked hypochromism in the ultraviolet region. The orderly double helical structure of DNA was found to play an essential rôle in this spectroscopic change. A high concentration of the interactant, low environmental ionic strength, low pH, and low temperature were beneficial in this interaction. The degree of hypochromism expressed by the integrated intensity and pKa of the benzacridines were found to be in parallel. The hypochromism of the interaction system, in which the carcinogenic benz[c]acridine derivatives took part, was larger than that of the system in which the noncarcinogenic derivatives were involved.

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Combination effect of antitumor agents, including cyclocytidine and cytosine arabinoside, was evaluated on the conception of pharmacological synergism and not of therapeutic synergism. Ascites sarcoma-180 and L-1210 leukemia were used as tumor systems. In sarcoma-180 system, combinations of cyclophosphamide plus cyclocytidine or cytosine arabinoside by simultaneous administration and cyclocytidine plus Daunorubicin or Vinblastine by alternate administration provided synergism. In L-1210 system, many compounds in combination with cyclocytidine or cytosine arabinoside in both simultaneous and alternate administrations provided synergism. Combination effect of agents was affected by the schedule of drug administration. It was found that the combination effect of drugs in one tumor system cannot be generalized to that in other tumor systems, even though equally effective doses of agents were administered in both tumor systems. Toxicity of cytosine arabinoside in combination with other drugs was affected by the schedule of administration. Compounds which provided synergism in simultaneous administration provided antagonism in alternate one. As a result, it was found that alternate administration of drugs is advantageous for the activity and diminution of toxicity to the host animal.

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Twenty-seven out of 31 cultured human cancer cell lines transplanted subcutaneously into nude mice produced solid tumors. Direct transplantation of surgical materials proved less successful. In 35 attempts, only 1 of 6 gastric cancers, 2 of 3 liposarcomas, and 1 of 3 osteosarcomas were accepted. No positive tumor formation was noted after the inoculation of 3 nasopharyngeal carcinomas or 1 breast cancer. None of lymphomatous neoplasms and leukemias produced any tumor in 12 and 3 attempts, respectively.
The histological and cytological characteristics of the tumors developed were studied with light and electron microscopes, in relation to the features in vitro and those of the parent tumors. Histological similarity of the tumors that developed in nude mice to their parent tumors was noteworthy. Serial transplantation was performed successfully in 9 cell lines and 2 tissues. Preservation of tissue construction ability and other differentiation abilities of in vitro cultured human cancer cells were ascertained in varying degrees.

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Effect of substitution of 5-position of cyclocytidine with fluorine on its antitumor activity in cultured cells was examined. 5-Fluorocyclocytidine was active against cultured L-5178Y cells similar to cyclocytidine. IC₅₀ of the compound was 0.054μg/ml. This compound inhibited thymidine incorporation into acid-insoluble fraction of the cells. Cell growth inhibition by 5-fluorocyclocytidine was reversed by deoxycytidine but not by thymidine and deoxyuridine. On the other hand, cell growth inhibition by 5-fluorouracil was reversed by thymidine and deoxyuridine. As a result, site of action of 5-fluorocyclocytidine was considered to be similar to that of cyclocytidine and not to 5-fluorouracil.

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Multinucleation of SV40-transformed mouse cells was induced by Cytochalasin-B as a result of inhibition of cytokinesis. Multinucleated cells with nuclei, whose number is other than that that can be obtained by raising the power of 2, were frequently observed. By simultaneous addition of Cytochalasin-B and cyclic dibutyryl-AMP to SV40-transformad mouse cells, multinucleation was fairly inhibited and the predominance of the cells with 2 or 4 nuclei was characteristic.
In case of normal mouse cells (BALB/3T3), addition of both Cytochalasin-B and cyclic dibutyryl-AMP lessened the number of nuclei in a cell compared with treatment of the cells with Cytochalasin-B alone.
These results suggested that cyclic dibutyryl-AMP inhibited multinucleation of cells treated with Cytochalasin-B and that the chemical regulated the division of nuclei in a cell to divide simultaneously.

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An immunosuppressive factor was purified from Ehrlich carcinoma ascites by the combination of ultrafiltration and Sephadex chromatography. The resulting product showed 50% reduction in the number of splenic plaque-forming cells in mice immunized with sheep red blood cells when as low as 50μg dose was given twice intraperitoneally before erythrocyte injection. The molecular size of the product was between 30, 000 and 100, 000, and it was relatively heat unstable.

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The number of cells producing the anti-Ehrlich tumor antibody (antibody against Ehrlich tumor cells) in the spleen of mice bearing Ehrlich solid tumor was counted by the modified Cunningham method, in which the sheep red blood cells were conjugated with extracted specific Ehrlich tumor antigen and used. The antibody-producing cells appeared 6 days after the subcutaneous transplantation of Ehrlich tumor cells and increased in number with a peak around the 15th day. Humoral anti-Ehrlich tumor antibody estimated by immune adherence gradually increased in the tumor-bearing mice, reached the maximum 15 days after the transplantation or later, and maintained the high level for a long period. The subcutaneous solid tumor grew and reached the maximum in weight around the 15th day and regressed thereafter.

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Short-chain fatty acids as sodium salts (pH 7.0) induced hemoglobin synthesis in cultured Friend leukemia cells (T-3-Cl-1) in concentrations of 0.5mM butyrate, 6mM isobutyrate, 2mM propionate, and 30mM acetate. Relative number of cells decreased by the addition of these fatty acids. Addition of 0.5mM butyrate to culture medium resulted in the highest hemoglobin synthesis as found by the extracted hemoglobin content by spectrophotometry.
The activity of δ-aminolevulinic acid synthetase increased on day 4 by the addition of any of these fatty acids into the medium. Hemoglobin synthesis was initiated on day 3 of culture by the addition of either butyrate or isobutyrate, and on day 4 by the addition of dimethyl sulfoxide. Hemoglobin accumulation reached the maximum on day 5 of culture in all cases when the fatty acid was added. Reasonably similar values were obtained for hemoglobin synthesis determined by either benzidine staining or hemoglobin quantitation. Formate, succinate, and citrate were ineffective for the induction.

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5-Nitroacenaphthene was proved to be mutagenic on TA100 and TA98 of Salmonella typhimurium with or without metabolic activation by S-9 Mix. A possible metabolite, 5-hydroxyacenaphthene, carcinogenicity of which had not been observed, was non-mutagenic on TA100 and TA98, with or without metabolic activation.

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Hemoglobin production was induced in culture lines of Friend virus-induced leukemia by addition of a human placental extract preparation (PLP). Two possible effective factors in PLP are discussed; a certain polypeptide of approximately 5, 000 in molecular weight and acetic acid. The latter appeared to act more effectively as an inducer.

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Gas chromatographic analysis was made for the content of N'-nitrosonornicotine in 10 kinds of Japanese tobacco products. Presence of N'-nitrosonornicotine was found in two products, doubtful in 5, and not detected in the remaining 3.

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Nagilactones extracted from Podocarpus species plants were tested for their carcinostatic activity. Among the five lactones, nagilactones E and D were most effective on Yoshida sarcoma cells in vitro, but nagilactone C was found most effective on CDF₁ mice bearing P388 leukemia.

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