Sensitivity of rat leukemia, DBLA-6, induced by N-nitrosobutylurea, to various antitumor agents was investigated. The tumor was proved to be sensitive to Vincristine, Daunomycin, 6-mercaptopurine, and alkylating agents, but efficacy of the compounds was decisively dependent on the combination of the routes of tumor inoculation and drug administration. The results of screening with this tumor gave important informations on the distribution of drugs in vivo and also the pattern of metastasis of the tumor.

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The initiation and growing points of DNA in partially synchronized L5178Y cells were studied by means of the M-band method and electron microscope autoradiography.
(1) A 10-min pulse of DNA with ³H-thymidine at the beginning of the DNA-synthesis (S) period of the ¹⁴C-prelabeled thymine-less cells revealed that the ³H label was found preferentially in the M-band as the nuclear membrane-DNA complex. The ³H label in M-band was partially chased with time and released into the supernatant free DNA fraction. After the chase, the M-band retained about 40% of the total ³H radioactivity which is the same amount as that of the ¹⁴C-prelabeled parental M-band DNA. The 3-hr label with ³H from the beginning to the mid-S period showed a similar result, demonstrating that the initiation sites are located on the nuclear membrane. The continuous labeling of M-band and the supernatant DNA indicated that two units of growing points migrated from a single initiation site on the nuclear membrane.
(2) The electron microscope autoradiography disclosed that ³H grains on thin sections after a 10-min pulse at the beginning of the S period were located mainly both on and near the nuclear membrane and in the perinucleolar region, but those in mid-S cells were found throughout the nucleus except in the nucleolus.
These data indicate that DNA replication in two directions is definitely initiated at the nuclear membrane and the growing points may migrate from it with the progress of DNA synthesis. The mechanism of involvement of nucleolus in the initiation of DNA replication is not yet clear.

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Differences in the urea-extractable antigens in mouse epidermis at several stages of carcinogenesis-hyperplastic epidermis, papillomas, and carcinomas-was investigated by employing immunodiffusion in agar with the urea antigens and immunoglobulins raised against these antigens. Normal mouse epidermis was found to contain urea antigens, some of which were the same as those present in hyperplastic epidermis. On the other hand normal epidermis contained urea antigens which were quite distinct from those of papillomas and carcinomas, and these normal urea antigens were not detected in hyperplastic epidermis. Hyperplastic epidermis, however, had distinctive urea antigens or ones which were present in much greater amount than in normal epidermis, papillomas, or carcinomas. The urea antigens of papillomas and carcinomas appeared to be the same and appeared to be common with some of the urea antigens of hyperplastic epidermis. Thus the conclusion is reached that the urea antigens of epidermis are altered at the stages of neoplasia between normal and hyperplastic epidermis, between hyperplastic epidermis and papillomas or carcinomas, and between normal epidermis and papillomas or carcinomas. In other words the urea antigenic changes which occurred at these different stages of carcinogenesis appeared to be abrupt and not gradual. An investigation of the urea antigenic modifications in epidermis at shorter periods of time after one or several applications of 3-methylcholanthrene, or after multiple applications of the carcinogen for several weeks or months (late hyperplasia) may reveal gradual changes in the urea antigens. Modifications in the types of epidermal cells (differentiating, degenerate, resting, resting in mitosis, and mitotic) at several stages of carcinogenesis did not appear to be related to the variations in the urea antigens.

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The examination of tumorigenicity, T and virus-specific transplantation antigens (VSTA) of adenovirus 12-transformed and tumor cell lines revealed that one cell line had both T antigen and VSTA, and one cell line had neither antigen. Two other cell lines had T antigen and might probably have VSTA to some extent. All tumors produced by implantation of these cell lines had T antigen. All of the test cell lines were tumorigenic and there was no correlation between VSTA and the intensity of tumorigenicity.

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Results of further experiments on a transplantable lymphocytic sarcoma of ddN mouse were described, including those of long-term observations in cell-free transmission and electron microscopic findings. The low rate of cell-free transmissibility, in spite of extremely rapid growth of sarcoma tissue grafts and consistent occurrence of C-type particles in sarcoma tissue, was taken to indicate that the virus associated with the sarcoma may be of weak oncogenicity.
The C-type particles were also detected in apparently normal lymph nodes of the sarcoma-bearing mice, of mice which had been injected neonatally with cell-free filtrates but failed to produce sarcoma, and also of old normal mice.

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The lethal effect of Mitomycin on cultured mammalian cells was studied by using mitotically synchronous and asynchronous HeLa cells. The cultures were treated pulsewise with the antibiotic for 1hr, and survivals were determined by the colony formation technique. Asynchronous cells revealed a composite type doseresponse curve and the use of synchronous cells suggested that two types of responses, exponential and sigmoidal, can be assigned to different stages of the cell cycle. The cyclic change in sensitivity was also observed during the cell cycle. The G1 and G2 periods were moderately sensitive, while the latter part of the S period was the least.
Effect of the antibiotic on DNA synthesis was also studied using DNA precursors. The maximum depression of DNA synthesis was found in the cells treated in G1 period. Behavior of the antibiotic-treated cells in the post-treatment generations was observed by time-lapse cinematography. The incidence of cell death mainly occurred after the first post-treatment cell division. These findings were useful for possible interpretation of the cell cycle dependence in cell sensitivity to Mitomycin.

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Incidence, morphology, transplantation, and virus studies on spontaneous tumors in the hamster colony are described. The incidence of spontaneous tumors in hamsters surviving over a year for their life-span was 6% (10/156); six malignant lymphomas, an osteosarcoma, a testicular fibrosarcoma, a hemangiosarcoma, and a salivary gland adenocarcinoma. Three of six malignant lymphomas showed a remarkable cutaneous involvement. Osteosarcoma had particles resembling C-type virus by electron microscopy. Testicular fibrosarcoma has not been reported previously in hamsters. These tumors were highly transplantable into young adult hamsters. Attempts to transmit these tumors by tissue extracts gave negative or indefinite results.

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Effect of X-irradiation was examined on the islet-forming ascites tumors of rats which grew in cell clumps floating in the ascites. Survival of irradiated cells was estimated from the delay of exponential growth of 10⁷ cells in the peritoneal cavity. In general, the islet-forming ascites tumor cells were more resistant to radiation than the free cells of the related tumor strains.
Concentration of non-protein, acid-soluble sulfhydryls in the islet-forming cells was found to be 6mM, higher than that in free cells. Eight mM of cysteamine, one of the radioprotectants, added to the cell suspension increased the intracellular, nonprotein sulfhydryls in Yoshida sarcoma cells and survival of irradiated cells to the level observed in the islet-forming ascites cells.

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Effect of X-irradiation on the liver of adult rats was studied. Parenchymal cell nuclei of the liver were microscopically distinguished from other littoral cell nuclei on smeared slides of isolated nuclei prepared in citric acid solution. Population analysis of parenchymal cells in the regenerating liver was made. An increase in parenchymal cells after partial hepatectomy was stepwise and suggested partially synchronous proliferation of the cells. The generation time and doubling time of the parenchymal cells calculated from the size of synchronous population and the growth curve were 36 and 67hr, respectively. X-irradiation on upper abdomen of rats was carried out 24hr prior to partial hepatectomy. The dose-dependent delay of the parenchymal cell proliferation was observed. Surviving fraction of irradiated parenchymal cells was assayed from the growth curve after partial hepatectomy. Parameters of radiation sensitivity, D₀, D_q, and extrapolation number were calculated as 250, 500 rads, and 7.5, respectively.

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The antitumor activity of cyclocytidine was examined in a variety of tumors. Cyclocytidine was active against adenocarcinoma-755, Nakahara-Fukuoka sarcoma, ascites sarcoma-180, Ehrlich ascites carcinoma, L-1210 leukemia, and C-1498 leukemia. Cures (60-day survivors) were observed at 500mg/kg/day×5 or more of the compound in the L-1210 system and therapeutic ratio was as high as 50, though that of other known antitumor agents tested, including 1-β-D-arabinofuranosylcytosine (Ara-c), was less than 12. Therapeutic index of cyclocytidine in the solid and ascites tumors was always greater than that of Ara-c. Cumulative toxicity of cyclocytidine was surprisingly low and LD₁₀ was 790mg/kg/day×5, whereas that of Ara-c was 82mg/kg/day×5. Cyclocytidine appears therefore to have advantages over Ara-c for clinical use.

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Comparative time-lapse cinematography of 12 human lympho-blastoid cell lines has permitted classification of cellular migratory morphology into pineapple, hand-mirror, and worm configuration. Only Burkitt lymphoma cells show little locomotive capacity. Evidence is presented indicating unique anatomical and functional properties of the uropod. Some interesting ultrastructural features are also included.

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Absence of acute toxicity in the carboxymethyl derivative of the antitumor yeast mannan was confirmed on more than 20 different lots in a dosage up to 20 times that of the total therapeutic dose against the subcutaneously implanted sarcoma-180 in mice (2, 000mg/kg×1, i.v.).

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Dextran sulfate decreased saturation density of tested three cell lines of cultured tumor cells without cytotoxic effect. Low molecular weight of dextran sulfate has no effect. Cells treated with dextran sulfate begin to grow after removal of the additive.

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Antitumor activity of haloacetylphenothiazine derivatives was studied against ascites sarcoma-180. 5-Oxide and 3-hydroxy derivatives were found to be more active than the mother compound, but therapeutic index was not improved by the substitution because of elevation in toxicity. Other substitutions reduced the antitumor activity of haloacetylphenothiazine.

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Maximum sublethal dose of 2, 2'-O-cyclocytidine reduced the rate of tumor growth and prolonged the survival period of the mice. It was suggested that a more satisfactory therapeutic effect may be expected by proper modifications of the administration method.

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The carcinogenicity of artemisia, horsetail, osmund, and ginkgo nuts, which are edible, was studied in inbred strain ACI rats. They were fed for 76 to 337 days with the pellets containing each plant material. Significant results suggesting the carcinogenic activity of these plants were not obtained.

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Examinations were made on whether the antitumor effectiveness is potentiated by the combined use of PC-B-45 and Mitomycin-C. It was concluded that PC-B-45 may be used in combination with Mitomycin-C to bring about an increased cytostatic effect on a tumor without producing further side actions.

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The metabolism of butyl(4-hydroxybutyl)nitrosoamine (BBN), a potent bladder carcinogen, was studied in rats. Three metabolites, butyl(3-carboxypropyl)nitrosoamine, butyl(3-carboxy-2-hydroxypropyl)nitrosoamine, and β-D-glucopyranosiduronate of BBN, were isolated and characterized from urine. The first metabolite was found to be as potent a bladder carcinogen as BBN.

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Hepatocarcinogenicity in dd mice of oral administration of α-, β-, γ-, and δ-isomers (500, 250, and 100ppm) of benzene hexachloride for 24 weeks was studied. All the animals (500ppm α-isomer) and 9 of the 20 mice (250ppm α-isomer) developed many tumors in the liver.

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In vitro infection of an established human lymphoblastoid cell line with Rauscher leukemia virus has resulted in the persistent release of abundant type-C virus particles along with a few EB virus particles. The human cell-passaged type-C virus elicited no leukemia when inoculated into newborn and weanling BALB/c mice.

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