The alleged modification of tumor cells by treatment in vitro with normal liver RNA, resulting in the greatly diminished cell transplantability (DeCarvalho and Rand, and Niu, Cordova, and Niu), was investigated in relation to the carcinostatic liver factor, which we previously reported.
It was found that the anti-tumor activity of our liver extracts was not affected in the least by digestion with pancreatic RNase, in spite of the fact that the RNA content of the extract was reduced almost to 10% by the RNase treatment. Without questioning the validity of the RNA effect claimed by other workers, therefore, we conclude that our carcinostatic liver factor may be totally unrelated to RNA.

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Histopathological studies on 23 cases of embryonal hepatoma are presented and summarized as follows:
1. The characteristics of neoplastic cells and neoplastic tissue of embryonal hepatoma were briefly described.
2. The structures which might be considered as compatible with the appearance of the so-called osteoid in the literature were found, variable in size, within the tumor tissue in 11 cases.
3. The conspicuous features were completely traced in process of their formation and were concluded to be hyalinously modified pattern of stromal connective tissue, just like localized cylindroma. There was no relation between bone and this hyalinous structure. It hardly seemed reasonable to put the cases submitted for the present study into the group of hepatic mixed tumor.
4. The presence of hematopoietic foci mainly composed of erythrocytic immature cells was observed within the neoplastic tissue in 9 cases.
5. These foci were considered to be formed in the dilated capillaries within the neoplasm by the influence of the neoplastic cells originating from hepatic cells.
6. Histogenesis of both particular findings, distinctly different from those of hepatoma in adult, in embryonal hepatoma was discussed with respect to tissue induction.

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The histochemical observation of aminopeptidase in the skin undergoing carcinogenesis induced by 20-methylcholanthrene was carried out in accordance with the method of Nachlas, Crawford, and Seligman.
1) Aminopeptidase became demonstrable in the subcutaneous connective tissue adjacent to proliferating epithelium, as the epithelium began to be hyperplastic and to invade peg-like.
2) This enzymatic activity was intense in the adjacent connective tissue as long as the invading property was observed, but diminished in the stadium of promoted carcinization with rich epithelial pearls.
3) Aminopeptidase was also demonstrated in some round cells and mast cells infiltrating in the subcutaneous connective tissue.

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The electronic structure of 2-acetamidofluorene and its related compounds is calculated using the frontier electron method, which has been established by the present authors, and compared with the chemical reactivity, formation of many phenolic metabolites, and carcinogenicity of these compounds.
The experimental positions of attack in fluorene, aminofluorene, and 2-acetamidofluorene are explained well by the frontier electron density, whereas the total π-electron deisity and localization energy fail to explain the experimental resuits of fluorene.
The formation of phenolic metabolites after ingestion of 2-acetamidofluorene is discussed by the frontier electron density of the molecule and it is found that the relative amounts of many ring-hydroxylated products are parallel to the order of the frontier electron density for radical attack. In view of this finding, the mechanism of ring hydroxylation of 2-acetamidofluorene is deemed to be radical in nature, and a similarity in reaction nature to other carcinogens such as aromatic hydrocarbons and azo compounds is pointed out.
The carcinogenicity of 2-acetamidofluorene and its related compounds is compared with theoretical index and an intimate correlation is found between the hepatocarcinogenicity of these compounds and the frontier electron distribution (approximate superdelocalizability) for nucleophilic attack at the 4-position. From this fact, it is supposed that the binding of the 4-position of these compounds with the nucleophilic (electron-rich) center in the body might be intimately connected with the formation of a tumor. This is an additional support of the previous supposition.
The nature of the binding receptor is discusssed in connection with the similarity between the theoretical conclusion and the property of binding proteins disclosed by experiments.
The sterical resemblance between the various kinds of chemical carcinogens is mentioned in regard to the hepatocarcinogenicity and the skin cancer-producing activity of aromatic hydrocarbons, azo compounds, 4-nitroquinoline 1-oxide, and 2-2-acetamidofluorene. Also attention is directed to the possible importance of the nitrogen atom in hepatocarcinogenic compounds.
Re-examination of the role of the K-region carbons in aromatic hydrocarbons is made in view of the sterical resemblance between the aromatic hydrocarbons and 4-nitroquinoline 1-oxide derivatives.

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The membrane potential of Ehrlich ascites tumor cell was determined with the use of a microelectrode under direct microscopic observation. Further, examinations were made on the effect of certain physical factors in environment upon the membrane potentials. The results thereby obtained are summarized as follows:
1) On the impalement of microelectrode into the cell, both positive and negative potentials were obtained.
2) The negative potential observed was invariably unstable and its time-course was characterized by a rather slow rise to the muximum and a gradual decay to a zero potential level, after staying in the maximum magnitude for only a very short duration. The mean maximum value of the potentials was -16.9±0.5mV.
The potential decreased markedly in the course of time after aspiration of the tumor cell.
The potential decreased significantly as the external hydrogen ion concentration increased.
The potential showed no significant change with the change of temperature within the range from 16°C to 37°C. However, it has a very small but positive temperature coefficient.
The increase of external potassium concentration caused the decrease of potential. Nearly a linear relationship could be found with the potential change on log [K]₀ of more than 32.0meq/L. The change in potential with a tenfold change in external potassium concentration was only 19.2mV.
3) The positive potential showed an intimate correlation with the tip junction potential of the microelectrode.

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An attempt was made to use cultured Yoshida sarcoma cells as a meterial for metabolic and morphological studies of tumor cells, and some preliminary experiments were carried out to provide a basis for it.
Yoshida sarcoma cells collected after 24 hours' cultivation in a mixture of equal amounts of bovine serum and Earle's balanced saline (S-E) were used for these studies. As the incubation medium for the measurement of glycolysis and respiration, a mixture of equal amounts of bovine serum and either Krebs-Ringer bicarbonate (S-K) or Krebs-Ringer Tris-buffer was used.
1) It was confirmed that Yoshida sarcoma cells could survive and continue to multiply in the medium S-K (in which the glycolytic activity was measured) for at least 48 hours.
2) Cultured Yoshida sarcoma cells retained their glycolytic and respiratory activities for more than 3 hours in such conditions. A considerably high rate of ³²P-incorporation into RNA-P or DNA-P by these cells was also observed during glycolysis and respiration.
3) The appearance of cultured Yoshida sarcoma cells remained normal and their mitotic figures were also found after the measurement of glycolysis and respiration.
4) The viability of cultured Yoshida sarcoma cells was proved by transplantation.
5) The effect of some agents was examined under the same conditions. In general, the degree of inhibition of ³²P-incorporation into RNA-P or DNA-P by the cells was greater during glycolysis than during respiration.
6) From these results, it seems that cultured Yoshida sarcoma cells are advantageous as a material for such studies.

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1) In order to clarify the mechanism of natural and acquired resistance of tumor cells to alkylating agents, comparative studies were made on cell permeability to methyl-bis(2-chloroethyl)amine N-oxide (MBAO) and methyl-bis(2-chloroethyl)amine (MBA), using the Yoshida ascites sarcoma, ascites hepatoma AH-130, AH-13, AH-602, AH-7974, and MBAO-resistant subline of AH-13.
2) On the basis of the known preferential affinity of alkylating agents to sulfhydryl groups, the permeability of tumor cells to alkylating agents was estimated by comparing the decrease of sulfhydryl content by contact with alkylating agents in intact tumor cells and in those whose cell membrane was previously destroyed. Sulfhydryl content was determined by the amperometric method.
3) Significant difference was not observed in the permeability to MBAO and MBA between the original AH-13 strain and its MBAO-resistant subline. The order of permeability in the original strains was the same in both MBAO and MBA. The order of decreasing permeability was AH-130, Yoshida ascites sarcoma, AH-13, AH-7974, and AH-602.
4) From the examination of cell permeability and that of sulfhydryl content reported in the previous paper, it was concluded that the non-protein sulfhydryl content and permeability of tumor cells play an important ròle in the mechanism of natural resistance of tumors to alkylating agents, while the development of acquired resistance to alkylating agents is chiefly due to an increase in non-protein sulfhydryl content.
5) It was further confirmed that the tumor cells which show a strong mutual adhesiveness are less permeable to alkylating agents.

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1) The effect of amino acid imbalance on protein synthesis was investigated both in tumor tissue and in normal tissues of rats. When one of the essential amino acids, tryptophan, was omitted from the amino acid mixture administered, protein synthesis in the normal tissues was about 60% of the control, whereas there was little effect on that of tumor tissue.
2) Evidence was obtained that ascites hepatoma AH-130 cells degrade plasma protein to amino acid components or small peptides prior to its utilization for protein synthesis.
3) The fact that plasma protein is utilized preferentially by the tumor tissue was reproducible using the solid form of hepatoma AH-130.

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