Two free-cell sublines of ascites hepatoma AH-130 were established. These sublines, AH-130F(G) and AH-130F(N), are composed of only singly isolated tumor cells without cell aggregates, "islands", differing from the original strain AH-130 which contains islands besides free tumor cells in the ascitic fluid. Furthermore, a clear difference was observed in chromosome constitution, ultrastructure, growth rate, and metastatic spread between these free-cell sublines and their original strain AH-130. AH-130-F(G) and -F(N) are also somewhat different from each other in biological properties. As to the susceptibility to antitumor agents, both of these free-cell sublines were more sensitive to nitrogen mustard N-oxide than AH-130. However, AH-130F(G) was rather resistant to Mitomycin-C, while AH-130F(N) was similar to AH-130.

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The pyridoxal phosphate content of the liver of tumor-bearing rats decreased steadily after ascites hepatoma (AH-130) cells were inoculated into the animals intraperitoneally. However, pyridoxal phosphate content of total cancer cells in the rat increased as the tumor cells proliferated rapidly. The activities of both pyridoxal kinase and pyridoxine phosphate oxidase decreased in the liver of tumor-bearing rats. This decrease in pyridoxine phosphate oxidase activity was restored to the normal level by the addition of a coenzyme, flavin mononucleotide, either in vivo or in vitro. Livers of rats fed on a non-protein diet showed as low a level of pyridoxal kinase activity as those of tumor-bearing rats. These results showed that the decrease of pyridoxine phosphate oxidase activity in the liver of tumor-bearing rats was due to a decrease in the concentration of its coenzyme, flavin mononucleotide. The decrease in pyridoxal kinase activity in the early stage of tumor formation was a factor causing the depletion of pyridoxal phosphate in the liver of tumor-bearing rats.
Both pyridoxal kinase and pyridoxine oxidase activities of tumor cells were about one-half of the activities of normal rat liver as calculated per gram of wet tissue.

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The cause of increase in the pyridoxal phosphate in tumor cells reported previously was examined. It was found that tumor cells did not have the ability to take up pyridoxine more rapidly than the host liver cells.
Indirect proof was obtained that tumor cells could take up vitamin B₆ from the tissues of the host.
Differences were found in the uptake of phosphorylated B₆ by tumor cells and by liver cells of tumor-bearing and normal rats. The tumor cells and the liver of tumorbearing rats could take up phosphorylated B₆ (pyridoxine phosphate and pyridoxal phosphate) without the elimination of phosphate, but normal rat liver could not. In normal rat liver, phosphorylated forms of vitamin B₆ were completely dephosphorylated by phosphatase.

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Change of the nicotinamide methylpherase activity of a rat liver in the state of tumor growth was studied. In the liver of tumor-bearing rat, nicotinamide methylpherase activity increased with the growth of the tumor. This increase is caused by various factors, such as the nutritional state of the host, hypophyseal and adrenocortical hormone. Toxohormone also has some effect on the change of nicotinamide methylpherase activity.

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The mitotic cycle of Yoshida sarcoma cells was studied by means of tritiated thymidine and autoradiography.
The duration of each phase of the mitotic cycle was estimated as follows:
(1) Early interphase (G1) lasted about 5.5-6.0 hours.
(2) Phase of DNA synthesis (S) lasted about 9.0-9.5 hours.
(3) Antephase (G2) was about 2.5 hours long.
The generation time of the sarcoma cells was determined as about 18.5 hours, which is almost in complete agreement with Freymann's result obtained by X-ray experiment.

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The increase of metastasis formation caused by selected carcinostatic agents is most pronounced when the host is treated with the agent before inoculation of tumor cells. In addition, present findings suggest that the adverse action of these carcinostatic drugs on the host-tumor relationship is more pronounced at a later stage of tumor development than initially. The effect of these agents on antibody formation was studied as a possible cause of this enhancement of metastases seen under suitable conditions. Nitrogen mustard N-oxide inhibits the production of antibodies against albumin injection and does so more effectively if the animal is preconditioned with the agent. Its action is still marked, however, even with treatment after albumin injection. Cyclophosphamide and Mitomycin-C also inhibit antibody production to albumin.
It has been demonstrated in human patients that carcinostatic agents depress the antibody response to diphtheria toxoid. A clinical case of regional perfusion is reported in which the drug injured the host tissues of the arm and the net effect of the chemotherapeutic agent was to increase the rate of tumor growth. In the cases of regional perfusion the response to challenge of a patient with diphtheria toxoid is increased over an untreated tumor-bearing patient and not decreased as is the case with the antibody response seen after systemic chemotherapy with a toxic drug.

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Toxohormone prepared from primary methylcholanthrene-induced fibrosarcoma, entirely free of bacterial contamination, markedly depressed the liver catalase activity in vivo. This confirmed the existence of toxohormone in tumor tissue itself, and disproved the allegation of Kampschimidt et al. that the liver catalase depression in tumor-bearing animals was due to bacterial contamination of the tumors. The results reported by Kampschmidt et al. were discussed in the light of the results obtained by the present work.

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RNA prepared from liver of rats given 4-dimethylaminoazobenzene [N-methyl-¹⁴C] (N-methyl-¹⁴C-DAB) 24 hours earlier was shown to be radioactive. The radioactivity was proportional to the specific activity of administered N-methyl-¹⁴C-DAB, The RNA was subjected to acid or alkaline hydrolysis, followed by ion-exchange column chromatography. The radioactivity was detected only in purine bases (adenine> guanine). Absorption spectra of the RNA showed that aminoazo dye is not bound to any detectable extent. The acid treatment of the RNA did not liberate any radioactivity in a volatile form.
These observations indicate that RNA is neither modified with aminoazo dyes nor with formaldehyde derived from N-methyl of DAB in acid-labile form. The methylation of guanine as occurring in the case of dimethylnitrosamine was also denied by the result of ion-exchange chromatography of the acid hydrolysate. The radioactivity of RNA was interpreted as being due to the incorporation of ¹⁴C-labeled C₁-compounds (formaldehyde and/or formic acid) derived from in vivo N-demethylation of the labeled DAB into adenine and guanine through the biosynthetic pathway, supporting the concept by Berenbom.

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4-Amino-6-octylresorcinol hydrochloride was further investigated for its antitumor activity on some transplantable mouse and rat tumors; Ehrlich ascites carcinoma, sarcoma-180, lymphatic leukemia SN-36, Yoshida sarcoma, and ascites hepatoma AH-13.
This chemical was effective not only in inhibiting the growth of both the ascites and solid forms of Ehrlich ascites carcinoma and sarcoma-180, but also in inhibiting the growth of ascites hepatoma AH-13. However, this compound did not show any antitumor activity on lymphatic leukemia SN-36 and Yoshida sarcoma.

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A transplantable AKR mammary tumor was successfully adapted to long-term tissue culture and the carcinoma cells have been serially passaged in vitro for 7 months. Ultrathin sections of the cultured cells revealed several cylindrical structures and many C particles unlike A and B particles initially observed in the original tumor.

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Subcutaneous injection of Mitomycin-C in rats bearing Yoshida ascites sarcoma results in a significant increase in the activity of DNase and acid phosphatase of Yoshida ascites sarcoma. The activity appeared to rise 3hrs. after Mitomycin-C injection, prior to the appearance of discernible morphological alterations.

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