Employing the Poisson distribution, we have investigated an approach to the occurrence of leukemia, using materials in the Tokai district during the period of 1962 to 1965. The occurrence of leukemia is in good accordance with the Poisson distribution and the critical test in order to evaluate the population vs. cases with defined critical region, at 95 or 99% of confidence limits, as computed to detect the areas with high occurrence. All 1108 units in the district were examined selecting 19 units at the 95% level and it was discussed whether the critical region thus defined was of validity in an actual phenomenon. Further inquiry is needed to set up the temporal-spatial unit, but this proved a good method for the evaluation of leukemia occurrence.

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Attempts were made to determine whether or not rat reticuloendothelial cells are capable of producing a cellular antitumor agent against Yoshida sarcoma in tissue culture.
An effective antigenic cell component was released into the culture medium from tumor cells after 3 days' culture in a Millipore diffusion chamber. The same cell components (sensitizing agent) were obtained from cultured medium of tumor cells after X-ray irradiation. Optimal doses of irradiation capable of releasing this agent ranged from 2, 000 to 4, 000R.
Sensitized bone marrow cells and abdominal macrophages were capable of producing an antitumor agent and inhibited growth of the tumor cells. Lymph node cells acquired antitumor activity only when mixed with the sensitized bone marrow cells or macrophages, but not when cultured with their supernatant fluid.
Immunological mechanism of in vitro production of the antitumor cellular agents in reticuloendothelial cells was discussed.

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Dissociated chick embryonic cells which had been precultured in monolayer and infected with Rous sarcoma virus (RSV) were tested in rotation cultures for their aggregability to elucidate the characteristic patterns of neoplastic transformed cells. RSV-infected chick cells produced aggregates with larger diameter in comparison with those from non-infected control cells.
A malignant tumor which appeared spontaneously in the mammary glands of the dd₁ mouse was dissociated and tested in rotation cultures for their aggregability. Mammary tumor cells formed some large aggregates, 0.8mm in diameter. This was in clear contrast with the absence of such large aggregates in control cultures of normal mammary gland cells. Changes in aggregation patterns shown in the neoplastic transformed cells were discussed in relation to changes accompanying differentiation.
Human cervical carcinoma, strain HeLa cells, were mixed with a variety of normal cells from chick embryos and tested in rotation cultures for their selective sortingout property in co-aggregates with normal cells from tissues of various embryonic origins. HeLa cells became sorted-out with embryonic chick liver cells and skin epidermal cells. On the contrary, HeLa cells formed chimaeric tissues interspersed with embryonic chick sikn dermal cells and limb-bud mesoblast cells. Types of original tissue from which HeLa cells were derived, and selective mechanism in metastasis of neoplastic cells were discussed.

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The free radical produced by stirring 3, 4-benzopyrene with albumin in aqueous solution was identified as 5-phenoxy radical of 3, 4-benzopyrene. The hyperfine structure of electron spin resonance (ESR) signal of the free radical did coincide neither with that of the cation radical nor with those of semiquinone radicals, but coincided entirely with that of the 5-phenoxy radical produced by oxidation of 5-hydroxy-3, 4-benzopyrene. Incubation of 3, 4-benzopyrene with liver homogenates fortified with co-factors gave the same free radical. Far less signals were observed when the co-factors were omitted or heated homogenates were used for incubation, and these results suggest that the phenoxy radical was produced via enzymatic reaction. The possible importance of the free radical production in carcinogenesis is pointed out.

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Some enzyme activities were measured with homogenates of livers from normal rats and from ones bearing Rhodamine sarcoma on the back. In addition, attempts were made to prepare, from the tumor tissue, substance capable of influencing the enzyme activities of mouse liver in vivo.
When rat livers were homogenized in a buffer containing Triton X-100, the catalase activity by the homogenates increased with increasing concentrations of the detergent; the activity increased by approximately 40% at 0.1% Triton X-100 but not further at higher concentrations. Both catalase activities, with and without Triton, decreased as the wet weight of tumor/body ratio increased, and were insignificant at the ratio of approximately 40%. The ratios in activity rate of the + Triton to +none were in most cases higher with tumor-bearing rats than with normal ones. The tryptophan pyrrolase activity was in most cases lower while the glucose-6-phosphate dehydrogenase activity was in some cases higher with tumorbearing rats than with normal ones. The cytochrome oxidase activity was hardly affected by the tumor/body ratio.
The precipitate obtained by centrifugation of the homogenate of the tumor, if injected into mice, influenced the levels of enzyme activities of the liver; the catalase and tryptophan pyrrolase activities decreased and the glucose-6-phosphate dehydrogenase activity increased, while the cytochrome oxidase activity was hardly affected. By injecting the precipitates obtained from the dorsal muscles and from the liver of normal rats, the catalase activity significantly decreased with the former but not with the latter. On the basis of minimum effective dose to influence liver catalase activity in vivo, the precipitate obtained from the muscles was approximately one-tenth as active as that from the tumor.
The precipitate from the tumor, if heated in a boiling water bath for 1 hour, still maintained in vivo-liver catalase-depressing activity but lost in vivo-tryptophan pyrrolase-depressing activity. Substance(s) capable of showing in vivo-liver enzyme-influencing activity were extractable with water from the precipitate from the tumor that had been washed with acetone and then with a mixture of chloroformmethanol (2:1 in v/v). When the extract was subjected to molecular sieve fractionation on a Sephadex G-25 column, the effluent fraction showed in vivo catalasedepressing and in vivo glucose-6-phosphate dehydrogenase-stimulating activities and the fraction eluted after inorganic salts present in the extract showed in vivo-tryptophan pyrrolase-depressing activity. When stored in a frozen state, the former fraction could maintain the activities without notable loss for months, whereas the latter fraction completely lost the activity within 1 week. The former fraction, if heated in a boiling water bath for one hour, completely lost the activities.

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Catalase extracted from the liver of normal rats with the aid of Triton X-100 was distinguished into several types having different isoleectric points (pI) by isoelectric fractionation with Ampholine carrier ampholytes devised by Vesterberg and Svensson.16) Of these types, four types were dominant in relative activity; "acidic" catalases had isoelectric points at 5.90 and 6.08, and "neutral" ones at 6.75 and 6.90±0.04 at 0-1°. The total activity of liver catalase decreased as Rhodamine sarcoma grew on the back of a rat; the activity of pI 5.90-catalase first decreased significantly, then followed by a decrease of the activity of pI 6.08-catalase, whereas the activity of neutral catalases rather increased until the ratio in wet weight of tumor to whole body reached approximately 20%. At higher ratios, the neutral catalases also decreased.
Catalase extracted from the liver of normal mice in the same way as from the liver of normal rats was also distinguished into several types, of which six major catalases had isoelectric points at 6.33, 6.40, 6.47, 6.53, 6.68, and 6.78±0.02 at 0-1°. When in vivo catalase-depressing substance prepared from Rhodamine sarcoma was injected into mice, the total catalase activity decreased; "acidic" catalases (pI 6.33-6.53) markedly decreased in activity, while "neutral" catalases (pI 6.74-7.08), which were little or not present in the liver of the control mice, increased in activity. Administration of the substance at any doses tested did not cause decrease in the activity of neutral catalases.
The dual changes of the acidic and neutral catalases by growth of the tumor was possibly caused by two different causes, one of which could be in vivo catalasedepressing substance produced by the tumor.

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Eight variant cell lines of the Yoshida sarcoma, characterized by island formation in ascites, were examined for their chromosomal constitution and DNA content. They differed from each other and from the original Yoshida sarcoma in the chromosome number and idiogram, i.e. they were different mutant cell lines with changed chromosomes. There was seen a general likeness in idiograms of three lines among the eight variants. A rough parallelism was present between the chromosome number and DNA content of the Yoshida sarcoma and its variants.

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Comparative differences were demonstrated in both cytological and biological characteristics of 12 different cell lines of ascites hepatoma. All these cell lines of ascites hepatoma were derived from isolated hepatoma nodules showing a histologically similar pattern, from the liver of a single female Donryu rat. Each cell line showed individual characteristics in ascitic feature, chromosome constitution, sensitivity to nitrogen mustard or X-ray, as well as in growth rate or invasiveness into the surrounding tissues. Multicentric cell origin of the primary hepatoma and a possible alteration in the cell population during successive transfer generations were discussed from an immunogenetic view-point.

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Pituitary prolactin levels, histological structures of ovary and mammary gland, and pattern of estrous cyle of the rat administered 7, 12-dimethylbenz[a]anthracene (DMBA) were little different from the control on 3, 5, and 10 days after administration of DMBA. These results suggest that DMBA had no effect on prolactin secretory capacity of anterior pituitary in rat.

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Oral administration of Cycasin, a glucosamine from Cycus revoluta nut, was given to 10 female rats of Sprague-Dawley strain. Two mammary cancers were detected; in one case, metastasis to the lung was found. Acceleration of the incidence of mammary cancer by Cycasin was suggested.

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Ten and/or 50% acetone solution of cigarette tar was painted on mouse skin for 1 year. Following kinds of tumors were induced: Malignant lymphomas, myeloproliferative disorders, lung tumors, liver tumors, skin papillomas and carcinomas, and ovarian cysts.

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