Tumor cells from rat hepatomas, AH-414 and AH-64A induced by azo dye, were preincubated in vitro with different dilutions of rabbit anti-hepatoma serum. Then 10⁶ cells were inoculated intraperitoneally into Donryu rats. After 6 weeks rats surviving without tumors showed resistance to further challenge with the same tumor cells. The ratio of the number of surviving rats after challenge with 10⁵ fresh tumor cells to those surviving after inoculation with serum-treated tumor cells was maximal using dilutions of antiserum of 1:240 with AH-414 tumor cells and of 1:400 with AH-64A cells.
Pooled sera from AH-64A-resistant rats, 2 weeks after a 3rd challenge with 10⁷ fresh tumor cells (RRS Lot III), had a cytotoxic effect on AH-64A cells at dilutions of up to 1:64 while sera isolated after the 1st or 2nd challenge (RRS Lot I or II) were weakly cytotoxic. The cytotoxicity was complement-dependent and stable on heating at 56° for 30min. RRS Lot III was active in the indirect immunofluorescence test.
Rabbit anti-rat liver serum did not induce resistance to AH-64A in Donryu rats although the dilutions used had the same immune adherence or cytotoxic titer as rabbit anti-hepatoma serum. Rabbit anti-Donryu rat hepatoma serum failed to induce resistance to cells of a 3-methylcholanthrene-induced sarcoma, AMC 60, of ACI/N rats. The correlation between the immune adherence and cytotoxic titers of the rabbit antiserum and its effectiveness in sensitizing the animals against tumor cells is discussed.

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Transplantation immunity of Donryu rats against ascites hepatoma AH-64A induced by azo dye was demonstrated by intraperitoneal injection of tumor cells pretreated with heteroantibodies in vitro. Hyper-immunity was induced by successive challenges with fresh tumor cells. The cytotoxic effect of the serum of resistant rats (RRS) against AH-64A tumor cells was not reduced after absorption with normal rat liver cells, but was slightly reduced after absorption with normal rat spleen cells. The cytotoxicity was absorbed completely with 5×10⁶ AH-64A tumor cells.
AH-64A, -B, -C, and -D are ascites hepatoma cell lines originating from a single Donryu rat. AH-64A and AH-64B cross-reacted with RRS while AH-64C and AH-64D, chemically induced DBLA-6 leukemia cells and normal lymph node cells of rats, did not react with RRS in indirect immunofluorescence and cytotoxicity tests.
A neutralization test was carried out by treating 2×10⁵ tumor cells with either RRS or immune spleen cell in vitro and then injecting them subcutaneously into irradiated rats (400R). It was found that 1:20 dilution of RRS protected the rats against AH-64A tumor cell growth while 1:40 and 1:80 dilutions of RRS caused some protection. A subcutaneous tumor mass developed after transplantation of tumor cells treated with RRS, but after about 2 weeks this began to decrease in size and disappeared completely within 6 weeks after transplantation. Treatment of AH-64A tumor cells with immune spleen cells at cell-to-cell ratios of 1:200 and 1:100 caused complete neutralization while normal spleen cells at a ratio of 1:200 had a slight effect. Treatment with immune spleen cells prevented tumor growth from the start. Most of the surviving animals were resistant to challenge with 1×10⁵ fresh AH-64A cells.
RRS was fractionated by cellulose acetate membrane electrophoresis and the amounts of β₁-and γ-globulin fractions were found to be 48 and 42% more than in normal rat serum. The immunoelectrophoretic pattern of resistant rat serum showed a stronger IgM precipitin line than that of normal rat serum.

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In spite of the recent progress in fluorometry, the measurement of carcinogenic hydrocarbons in tissue has been carried out with insufficient accuracy because of the presence of substances in the tissue interfering with fluorometry. Such substances, autofluorescent and quenching substances, were removed from ethanol extracts of the tissue by Sephadex LH-20 column chromatography. By elution of the column with ethanol, the interfering substances appeared in fractions of the void volume for 3-methylcholanthrene chromatography. In addition, the excitation wavelength for autofluorescent substances was found to be far from that for 3-methylcholanthrene, for which the excitation was at 300nm and the luminescence was measured at 400nm.
Chromatographic separation of 3-methylcholanthrene and its metabolites in the bile and feces obtained from rats was done and only one peak consisting of conjugated metabolites was found on the chromatogram of the bile, though three peaks consisting respectively of the parent hydrocarbon with some metabolites, nonconjugated metabolites, and conjugated metabolites were observed in the feces
Total amounts of 3-methylcholanthrene and its metabolites, though the majority of the fluorescent substances were the parent hydrocarbon, in the lung and other organs were mearsured at various intervals after an intrabronchial application of 1 or 5mg of 3-methylcholanthrene in Freund's incomplete adjuvant to rats. Preservation of the hydrocarbons in the treated lung was confirmed and this was considered to take part in the enhanced occurrence of epidermoid tumors in the lung.

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Approximately 350 amino acid derivatives were synthesized and tested for antitumor activity in four tumor systems. The effect on life prolongation and tumor growth was examined using mouse leukemia SR-61, Ehrlich ascites carcinoma, ascites sarcoma-180, and rat ascites hepatoma (AH-60C). Among these 350 derivatives, 29 compounds were found to be significantly effective in prolongation of the median life-span and inhibitory effect on tumor growth in the primary screening. Among these 29 compounds, the following five compounds were found to possess potential antitumor activity: N-(2-Naphthalene)sulfonyl-DL-tryptophan (A-91), 2-naphthylaminomethyl-γ-aminobutyric acid (A-144), N-ethylcarbaminomethyl-L-isoleucine (A-145), N-9-fluorenylacetyl-L-phenylalanine (A-192), and N-propionyl-L-valine (A-195). These five compounds were active in prolongation of the life-span of mice bearing Ehrlich ascites carcinoma and in the inhibition of the cell growth. Some of these amino acid derivatives inhibited biosynthesis of macromolecules, DNA, RNA, and protein, in tumor cells.
These results suggest that the site of action of the five amino acid derivatives appears to result from the inhibition of macromolecules and another unknown mechanism.

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Administration of N-butylnitrosourea (BNU) induces leukemia in thymectomized C57BL/6J and C3Hf/Bi mice with almost the same high frequency as in non-thymectomized mice. Thymectomized and BNU-treated (C3Hf/Bi×CBA/H-T6T6) F₁ mice receiving neonatal thymus tissues from C3Hf donors developed leukemias with or without marked enlargement of the grafts. The origin of leukemic cells was analysed by T6 marker chromosome and thymus allo-antigen theta in this hybrid system.
Cells from leukemia with enlarged thymus grafts possessed the θ-antigen detected by cytotoxicity tests. Cells from leukemia without thymus involvement had no θ antigen. The leukemic cells arising at the site of thymus grafts were derived from the graft itself (C3Hf) or from the host (C3Hf×CBA/H-T6T6) F₁ cells, most probably bone marrow cells which are repopulating into the graft. When the mice were treated with BNU after the lymphoid elements in the grafted thymus had been replaced by host cells, leukemia mainly composed of host-origin cells developed. Leukemia in which neoplastic cells in the thymus grafts were of donor origin and those in other hematopoietic tissues were of host origin was found not infrequently. The present results mean that the target cells in BNU leukemogenesis are distributed within and outside the thymus and that some leukemias are of multifocal tissue origin.

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The prerequisit of reduction for activation of Mitomycin-C and unstability of its reduced form suggested investigation of the possible formation of free radicals (semiquinone forms) of a series of quinone-containing anticancer chemicals in vitro. The ability of rat-liver microsomes to initiate sulfite oxidation in the presence of NADPH was markedly enhanced by the addition of these chemicals. This strongly suggests that these chemicals participated in the process in the form of reactive free radicals. The reaction was specific for NADPH. Carbazilquinone was unique among others in that NADH can replace NADPH and its higher ability to initiate sulfite oxidation. Microsomes from Ehrlich ascites and AH-109A hepatoma cells were also effective, though to a lesser extent than those from rat liver on a protein basis. Generation of free radicals, though their biological significance is not clear at present, may be deemed an inherent chemical property of these chemicals.

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For the purpose of inducing experimental lung cancer, carcinogens were applied into the bronchus of the rabbit. Experiments were divided into 2 groups, I and II, depending on the methods of application of chemical carcinogens into the bronchus. In Experiment I, carcinogens (4-nitroquinoline 1-oxide and/or 3-methylcholanthrene) in rabbit plasma or distilled water were instilled in the lower bronchus and, in Experiment II, 3-methylcholanthrene in Tween 60 was swabbed on the main bronchus through a specially made bronchoscope. (1) In Experiment I, 163 of 366 rabbits having received over 4 instillations of the chemical carcinogens and surviving for more than 30 days developed lung cancer. However, in Experiment II, only 2 of 65 rabbits surviving more than 60 days developed lung cancer. (2) In Experiment II, 39 (60%) of 65 rabbits were found to have developed amyloidosis. However, in Experiment I, 86 (approx. 23%) of 366 rabbits developed amyloidosis. Amyloidosis was observed to occur in the kidneys, spleen, liver, and adrenals in both experiments.

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The growth of transplanted carcinomas originating in a gonadectomized C3H male mouse was much faster in intact male mice than in intact or gonadectomized females. Administration of 17β-estradiol or testosterone propionate in pellets resulted in a marked acceleration of the carcinoma growth in intact female mice. Progesterone and deoxycorticosterone acetate exerted no influence on the carcinoma growth. Neither gonadotropins (pregnant mare's serum gonadotropin plus human chorionic gonadotropin) nor ACTH accelerated the growth. In contrast, cortisone acetate in pellets markedly reduced the growth rate of carcinoma transplants when administered simultaneously with the transplantation. Cortisone acetate also arrested the growth-accelerating effect of 17β-estradiol on transplanted carcinomas.

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When Yoshida sarcoma cells were inoculated subcutaneously into the left footpad of Donryu rats, the weight of the left lumbar lymph nodes increased proportionally to the number of the inoculated cells. Resection of primary implants the day after the inoculation of 5×10⁶ cells failed to rescue the host rats and they died of metastases within 12 days. There was no significant difference in mean survival days between the rats (8.9 days) whose primary implants were resected on the 5th day after inoculation and the control rats (8.6 days) bearing the primary implants.
Mitomycin-C, a highly effective drug against Yoshida sarcoma cells, produced many long survivors (over 30 days) in the treated rats whose primary implants were resected the day after inoculation, but failed to do so when resected 5 days after inoculation. All of them died of metastases within 20 days.
Based on these results, this present system is discussed for its usefulness as a screening and evaluation model for antitumor agents.

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1) When nuclear fraction prepared from Rhodamine sarcoma (sarcoma nuclear fraction) was injected into mice three times every 24hr, the catalase activity of the liver decreased to one-third of the original activity.
2) By the injection of sarcoma nuclear fraction into mice, the catalase activity with the soluble fraction from homogenates of the liver decreased more significantly than that with the particulate fraction from them.
3) Immunological titration proved that the decrease of catalase activity in the liver of mice injected with sarcoma nuclear fraction was brought about by decrease in the amount of catalase protein.
4) In the mice, whose liver catalase activity had been irreversibly inhibited by injection of 3-amino-1, 2, 4-triazole, the initial rate for the restoration of the liver catalase activity was significantly slowed by further injection of sarcoma nuclear fraction.
5) When the inhibitor of catalase biosynthesis, allylisopropylacetamide, was injected into mice, the activity level of the liver catalase decreased. The extent of decrease by the injection of the inhibitor was slightly lower than that by the injection with sarcoma nuclear fraction, which was almost the same as the extent of decrease by the injection of sarcoma nuclear fraction plus allylisopropylacetamide.
6) It is conceivable that the catalase biosynthesis in the liver was inhibited by the injection of sarcoma nuclear fraction in almost the same manner as by the injection of allylisopropylacetamide. However, it is not certain whether the degradation of liver catalase was slightly stimulated by the injection of sarcoma nuclear fraction.

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In the hexokinase isoenzyme pattern on the cellulose acetate membrane electropherogram of the supernatant fraction of Ehrlich ascites tumor cells, a band was detected moving slower than the common Type I isoenzyme. This band was assumed to be due to the presence of a variant of the hexokinase isoenzymes in the cells, since such a band was not detected in the normal tissues, and a change in the intensity of the band in response to a sulfhydryl agent, etc., differed from that of the common Type I and II isoenzymes. The band was also found in AH-7974 and MH-134 ascites tumor cells, in spite of the difference in the species of the tumor-bearing animals and originating tissue.

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Two transplantable strains of adenocarcinoma were established from the carcinomas of the colon and rectum induced by infusion of N-methyl-N'-nitro-N-nitrosoguanidine in inbred ACI/N rats. The tumors are solid type, not converted to ascitic form yet, either papillary or tubulo-papillary adenocarcinomas, and particularly grow slowly, showing 2 to 4 months of survival in animals transplanted subcutaneously. Besides histological resemblance of the tumors to human adenocarcinoma of the large intestine, marked mucin production in tumor was noted in one of these strains.

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Survival period of rats bearing ascites hepatoma, AH-60C, which was less sensitive to bis(3-methylsulfonyloxypropyl)amine (864-T) and 6-mercaptopurine each alone, was prolonged by the combined treatment with these two agents. Two or 3 rats in groups of 12 animals each survived over 60 days by this combined therapy beginning from day 0. In AH-109A, which was insensitive to 6-mercaptopurine, the group treated with this combination did not survive longer than that with 864-T alone.

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Hepatocellular mitoses are scanty or absent in livers of animals in the late stages of growth of Ehrlich ascites tumour. The challenge of CCl₄-induced liver necrosis in such animals is, however, met by a massive hepatocellular mitotic response. Thus general debility of the animal, imposing a block on liver cell division, cannot be an explanation for the cessation of tumour-associated liver cell mitosis in late-stage tumour-bearing animals. Possible explanations for the pattern of hepatocellular mitosis in relation to duration of tumour growth are discussed.

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The relationship between the cell viability and frequency of cell with Epstein-Barr virus (EBV)-associated nuclear antigen was investigated in cultures of 4 EBV-carrier lymphoblastoid cell lines, by means of anti-complement immunofluorescence. The percentages of viable cells and those of EB nuclear antigenpositive cells were close to each other in the entire course of cultivation of all cell lines. This leads to a conclusion that absence of EB nuclear antigen in a given cell population should be evaluated in relation to cell viability.

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Experimental gastric adenocarcinomas induced by N-methy-N'-nitro-N-nitrosoguanidine (MNNG) were often found to be well-differentiated and hematogenic metastasis of the carcinoma cells is hardly observed. A case of poorly differentiated adenocarcinoma of dog stomach induced by MNNG with hematogenic metastases is reported for the first time.

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In order to investigate the histogenesis of lung cancer, normal bronchial epithelium was cultured. Outgrowth of epitheloid cells with pavement-like arrangement was observed, some of which had cilliae showing active movement. Secondary culture has not been successful yet.

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Production of α-fetoprotein in cultured ascites hepatoma AH-66 cells increased by extracellular addition of cyclic AMP and cyclic dibutyryl-AMP. Increased production of α-fetoprotein in AH-66 cells also occurred to some extent when higher concentrations of sodium butyrate and 5'-AMP were used for the treatment.

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