The rôle of virus in leukemogenesis by a potent chemical carcinogen, 20-methylcholanthrene, in RF mice was investigated.
1) The incidence of leukemia induced by 20-methylcholanthrene paintings was 88% (48% of lymphocytic leukemia and 40% of myelogenous leukemia). The incidence of spontaneous leukemia in these RF mice was 0.7%, 2 out of 300 mice.
2) Inoculation of cell-free filtrate from 20-methylcholanthrene-induced lymphocytic and myelogenous RF mouse leukemia into newborn mice of the same strain produced lymphocytic leukemia in 4 of 13 inoculated mice. Serial cell-free passage of the leukemia was also successful and the third passage is now in progress.
3) Virus particles were observed by electron microscopy in the lymph node of 20-methylcholanthrene-induced leukemic mice.
From these facts, the authors conclude that the chemical carcinogen induces leukemia by activating a latentvirus naturally resident in the RF mice.

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Fundamental conditions suitable for the cultivation of MN-lymphosarcoma cell were assayed and the results were presented as follows:
1) Static culture was a more suitable method than roller tube culture.
2) The culture in initial pH 7.6 was found to be optimal.
3) Continuous cell multiplication was obtained with the inoculum size of over 100, 000 nuclei/ml/tube.
4) 0.4% Lactalbumin hydrolysate in the medium was found to be optimal.
5) The optimal concentration of yeast extract was determined as 0.08% in the medium.
6) Vigorous propagation of the cells was obtained with a medium containing 40% bovine serum.
7) The cell multiplication was not sustained in the medium in which dialysed bovine serum was substituted for nondialysed one.
8) The cell growth was accelerated markedly in the medium supplemented with dialysed bovine serum after the addition of hexestrol, a synthetic estrogen, to it.

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Three tissue culture strains were established from MN-lymphosarcoma cells originally carried intraperitoneally in NA₂ female mice. They have been maintained by serial subcultivation for more than 16 months. There were no apparent differences except for cell size in morphological features among the 3 strains. When inoculated into mice intermittently after prolonged maintenance in vitro, strain No. 1 and No. 3 cells induced ascites tumor formation and a mortality of 80-100%. Strain No. 2 cells seemed to have lost their transplantability. When the cultured strain No. 1 cells were carried serially by mouse-to-mouse passage, the mean survival time, which was longer in the first passage, was shortened within a few generations to that of the original mouse-to-mouse passage. This result might be discussed on the hypothesis of a gradual transformation of cells for adaptation to growth in altered environment.

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Asparaginase activity in the liver of rats fed 4-dimethylaminoazobenzene (DAB) was studied semiqualitatively by using paper electrophoresis and was represented in a pattern taking into consideration the length of migration of the enzyme on the paper. Two prominent peaks were found in the pattern in the case of normal liver in the digests of pyruvate and phosphate added. One was faster migrating part (peak I) and the other slower (peak II). The peaks I and II were confirmed as Greenstein's asparaginase I and II, respectively, by analysing the activation effect of phosphate and pyruvate, and more by heat treatment. The pattern of cirrhotic liver was almost similar to that of normal liver but that of hepatoma, on the contrary, kept peak II even low, and no sign of peak I. The pattern of rats fed DAB without interruption for 4 weeks lost peak I completely, but kept peak II as in the case of normal liver. After 11 weeks of experiment peak II became very low and the pattern resembled that of hepatoma.

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The activity pattern of liver asparaginase was investigated by paper electrophoresis, before and after feeding amino azo dyes. The dyes tested were 3'-methyl-4-dimethylaminoazobenzene, o-aminoazotoluene, and 4-aminoazobenzene. These chemicals were incorporated in the diet of rats on the same molar level as 4-dimethylaminoazobenzene reported in an earlier paper. 2-Acetamidofluorene was also used in the present experiment.
Some of the chemicals affected the pattern of asparaginase activity, while the others gave no effect.
Similar examinations were made with fetal and regenerating rat liver. The pattern of the former resembled that of hepatoma and of the latter to that of normal liver.
Similar experiments made with mice fed with 4-dimethylaminoazobenzene in their early experimental days showed that the asparaginase activity was stronger and resistant to the chemical than in rats.

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Ehrlich ascites tumor, DAB hepatoma of rat, and various human cancers contained a nitrate reductase which has not been found in normal tissues. The activity was also seen in rat liver for a time during its regeneration. The enzyme reduced nitrate, when reduced phenosafranine, DPNH, or succinate-succinoxidase system was supplied as a hydrogen donor. The sensitivity to inhibitors suggested the presence of iron as a metal constituent in the enzyme.

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Quantitative studies on the fractionation of carcinostatic liver factor were carried out employing bovine liver as material, and the carcinostatic activity was found to be assayable quantitatively and fairly clear dose-response relationship was obtained.
The specific carcinostatic activity of the boiled supernatant of the aqueous liver extract is increased almost 3 times by the successive procedures of ethanol extraction, dialysis against water, and water elution through cation exchange resin (Amberlite CG-120). Total activities are well recovered throughout these procedures.
The general properties of this liver factor, which is heat-stable, ethanol-soluble, dialysable, and anionic in nature, are discussed in connection with those of the cytolytic and/or growth-inhibiting substances in the tissue extracts previously reported by other investigators.

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MN Ascites sarcoma T strain was continuously propagated in vitro for more than 8 months. The growth of culture was maintained by LY medium supplemented with 20% ox serum. The culture vessels were mounted on a rotatory drum rotated at 10-12 revolutions per hour. The cells grew in suspension. Therefore, subcultures were made easily by transferring culture medium in which the cells were suspended. The inoculation of cultured cells caused enoromous accumulation of ascites and death of the animals. Metastatic lesions were formed which resembled those of the original tumor cells.

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MN Ascites sarcoma cells (T strain) maintained by serial transfers in female mice (animal line) and those maintained by continuous culture in vitro (culture line) were compared with respect to their virulence and chromosomal constitution.
1) The host of different sex showed different responses to the inoculation of either cell line.
2) Virulence to females decreased markedly after cultivation in vitro as evidenced from marked prolongation of survival time. The survival time of males was not changed significantly. Inoculation of culture line allowed females to survive till the cells increased to a much larger population than tolerated by the same hosts inoculated with the animal line.
3) Histotrophism of the culture line was lower than that of the animal line. The difference was seen in males and in females.
4) These two lines showed difference in their chromosomal constitution. The culture line was hyperdiploid, whereas the animal line was hypodiploid. The chromosomes of culture line showed greater morphological variations and the individual chromosomes as well as chromosome sets of the culture line were distinct from those of the animal line. Gradual changes of chromosomes were observed during long-term propagation in vitro.
5) Possible mechanisms for cytological changes were discussed. Rearrangement or reconstruction of chromosomal materials was suggested. It was impossible to correlate the alteration of virulence with changes of particular types of chromosomes.

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Survival time of the host mice, multiplication of the tumor cells in ascites, hemorrhage into ascites, accumulation of ascitic fluid, tumor cell infiltration into abdominal viscera of mice, and solid tumor formation in the lung of mice by intravenous tumor cell inoculation were compared among hyperdiploid, hypertriploid, and hypotetraploid lines of Ehrlich ascites tumor.
Malignancy of these tumor lines was found to increase in the order of the hyperdiploid, hypotetraploid, and hypertriploid tumor in terms of the smallest size of inoculum required for giving rise to the critical survival time of mice, which conceivably expresses the magnitude of malignancy.
None of these properties was concluded as directly relating to the death of the host. Correlations between these propereties and malignancy of the tumor was discussed in detail.

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In the course of an investigation on acquired resistance of Yoshida sarcoma to antitumor agents, there was a need for quantitative estimation of resistance grade. The present paper deals with the introduction of in vivo and in vitro methods for the determination of resistance index, out of which two methods employing the primary culture of tumor cell suspension seem to be very convenient and exact. The comparison of the data obtained by these methods was also made.

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Deoxyribonucleic acid (DNA) content was quantitatively studied by means of Feulgen-microspectrophotometry in tumor cells derived from three cases of gastric carcinomas and 18 cases of their metastatic tumors. In two of the gastric tumor cases the DNA content in tumor cells of the primary tumors was at almost the same level or nearly similar to that of metastatic tumors. In the other case the primary tumor showed a DNA value lying in a hyperdiploid range, while its three metastatic tumors showed a hypertetraploid value.

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To study further the causal relationship between severe benign liver damage and bladder neoplasms, liver injury was produced in male rats by subcutaneous injections of carbon tetrachloride or feeding of an ethionine-containing synthetic diet. All rats were then fed synthetic diets containing 2-acetamidofluorene (AAF) or AAF and indole for up to 12 months. The apparent hepatotoxicity of AAF was enhanced and all rats fed AAF alone after pretreatment with carbon tetrachloride died of severe hepatic damage within 66 days without tumors. Yet in rats fed AAF and indole, the previously demonstrated protective action of indole against hepatotoxicity of AAF was also enhanced providing a much more striking demonstration of protection by indole than was seen in the previous experiment. Pretreatment with carbon tetrachloride decreased tumor incidence in all sites. The ethionine feeding preceding AAF-indole treatment also failed to increase tumorigenesis in the liver and bladder. Therefore, the present experiments did not confirm the postulated enhancement of AAF carcinogenicity by prior hepatic damage induced by carbon tetrachloride or ethionine pretreatments. On the contrary, carcinogenic action of AAF may even have been diminished, at least in presence of carbon tetrachloride-induced cirrhosis where a great diminution was observed.

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Development and growth of a tumor were examined when the mesenchymal tissue was damaged by administration of drugs or by intervention of the nervous system, in order to clarify the relationship between the mesenchymal system and the tumor.
When cortisone or Trypan Blue was administered to a rat, foci of the lung caused by the cancerogenic substance tended to undergo malignant change more intensely than that of the control group. When the striate body of a rat was destroyed by electrocoagulation or injection of a mercuric chloride solution, the growth of subcutaneously implanted Yoshida sarcoma was accelerated. Skin tumor induced by 9, 10-dimethyl-1, 2-benzanthracene developed more frequently on the leg of the mouse after the sciatic nerve was cut. Incidence of skin tumor became greater when cortisone or Trypan Blue was administered in addition to cutting of the sciatic nerve.
These experimental results showed that the development and growth of a tumor were promoted by damage of the mesenchymal tissue, assumedly due to the weakened defensive response of the living body against invasion of a tumor.

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Experiments were performed using nitrogen mustard-sensitive and -resistant Yoshida sarcoma cells, on the change of drug resistance of tumor cells.
DNA extracted from nitrogen mustard-resistant cells showed a positive diphenylamine reaction and had ultraviolet absorption maximum at 260mμ, showing the typical spectrum of deoxyribonucleic acid.
When nitrogen mustard-sensitive cells were incubated with DNA extracted from nitrogen mustard-resistant cells, the sensitive cells obtained resistance against nitrogen mustard through transformation of tumor cells. These tumor cells showed a complete resistance against 0.2mg/kg of nitrogen mustard and an incomplete resistance against 0.5mg/kg of it. The original resistant cells showed a resistance against 0.65mg/kg of nitrogen mustard. Therefore, the acquired resistance through transformation was lower than the original resistance.
The conditions required during incubation to cause sufficient change was as follows: Concentration of DNA, 0.5mg/ml; amount of DNA corresponding to one sensitive cell, 2.38×10^-5μg; incubation time, more than 60 minutes; optimal temperature, 37°. Coexisting ascites promoted transformation.
The resistance which was invested to the sensitive cells by incubation with DNA extracted from Yoshida sarcoma cells resistant to nitrogen mustard was maintained through successive transplantations and inherited from cell to cell.

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