The effect of persistent infection with paramyxovirus, HVJ (hemagglutinating virus of Japan), on the transplantability of cultured tumor cells (THEL cells) was examined in hamsters. THEL-HVJ cells, HVJ-carrier cells without cytopathic effect, showed a lower transplantability than THEL cells. This lowered transplantability of THEL-HVJ cells was enhanced in X-ray-irradiated (550R) hamsters. THEL-HVJ tumors formed by transplantation of a large number of cells regressed occasionally. Spleen cells from the THEL-HVJ tumor-bearing hamsters were adsorbed on THEL-HVJ cells and showed marked colony inhibition in vitro. The cells from normal or THEL tumor-bearing hamsters showed a weak colony inhibition. The adsorption of spleen cells on THEL cells and their colony inhibition of the same cells was scarcely observed.
Thus, these THEL-HVJ cells appeared to have the cell surface reactivity immunologically different from that of non-infected, parent cells (THEL cells). These differences might be closely correlated to the lowered transplantability of THEL-HVJ cells in hamsters.

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Subcellular localization of gs antigens for avian leucosis-sarcoma complexes was studied in mouse ascites sarcoma cells (SR-C3H/He cells) induced by Schmidt-Ruppin strain of Rous sarcoma virus. Two techniques were used; (i) indirect fluorescent antibody staining of the cells and (ii) complement fixation after systematic subcellular fractionation. It was found that SR-C3H/He cells continue to produce the gs antigens even 6 years after the establishment of the ascites cell line. The gs antigens were exclusively localized in the cytoplasm and found in a high concentration in both the supernatant (soluble fraction) and the microsomal fraction, but could not be detected in the nucleus, mitochondria, or plasma membranes.

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1. The enzymic oxidation of benzidine compounds by rat liver and beef heart cytochrome a was studied spectrophotometrically.
2. The mitochondrial oxidation of 3, 3'-diaminobenzidine was inhibited by cyanide, azide, and heat-treatment for 10min at 60°, while it was activated by the addition of cytochrome c. Although approximately 70% inhibition was observed when 10mM succinate was added, activity was restored after the further addition of malonate, which suppressed succinate dehydrogenase activity. Oxidation was confirmed to be due to cytochrome oxidase activity.
3. 3, 3'-Diaminobenzidine was successfully oxidized by beef heart cytochrome a, and stimulated by the addition of cytochrome c. The molecular activity of cytochrome a alone was estimated as 0.72 (mot of diaminobenzidine/mol of cytochrome a/min). The rate of oxidation by cytochrome a and c decreased in the order of 3, 3'-diaminobenzidine, 3, 3'-dimethoxybenzidine, 3, 3'-dimethylbenzidine, and benzidine.
4. A possible relationship between the cytochrome c-reducing ability of benzidine derivatives and their carcinogenicity is discussed. Potent carcinogens, 3, 3'-dichlorobenzidine and p-aminobiphenyl could not reduce cytochrome c; and benzidine, 3, 3'-dimethylbenzidine, and 3, 3'-dimethoxybenzidine were shown to be moderate reductants. 3, 3'-Diaminobenzidine, a mild carcinogen, markedly reduced cytochrome c. Carcinogenic diphenylamine did not react with cytochrome c, but p-aminodiphenylamine quickly reduced it, suggesting that it has little or no carcinogenicity.

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Distribution studies were carried out in both the normal and the solid sarcoma-180-bearing mice with the carboxymethyl derivative of the antitumor yeast polysaccharide, which was labeled either uniformely with tritium or exclusively in carbonylcarbon of the carboxymethyl groups with carbon-14.
An outstanding uptake of the radioactivity was demonstrated in the liver and the spleen throughout the period of observation after a single intraperitoneal administration. The solid tumor also showed a moderate uptake in the order of one-tenth of the total radioactivity recovered in all the organs examined. These results were discussed in relation to the possible mechanism of action.
The urinary recovery of the radioactivity during a 24-hr period was 30% in the normal and 20% in the tumor-bearing group. The rate of urinary excretion decreased with time after administration, probably due to a shift in molecular weight composition towards a higher direction. A peak of the blood level in normal mice was observed 3hr after the intraperitoneal injection, while that in the tumorbearing mice was found after 6hr.
No substantial difference was found between two types of the labeled derivative in either the distribution pattern or the rate of urinary excretion.

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The formation of DNA pyrimidines by de novo synthesis (de novo pathway) and from preformed precursors (salvage pathway) was studied.
In regenerating liver, labeled precursors for both the de novo pathway and the salvage pathway were incorporated into DNA much more than in normal liver. Synthesis via the de novo pathway preceded that through the salvage pathway during liver regeneration, and the uptake of labeled deoxythymidine triphosphate into DNA preceded that of labeled orotic acid and thymidine during liver regeneration.
In marrow cells the incorporation into DNA of labeled precursors for the de novo pathway was very low, while that for the salvage pathway was quite high. In marrow cells, in which the blood-making function had been increased by blood-letting, the incorporation of labeled precursors for the salvage pathway increased considerably, while that for the de novo pathway did not. The incorporation of labeled deoxythymidine triphosphate preceded that of labeled thymidine in these cells.
Yoshida sarcoma cells showed the same pattern of incorporation as marrow cells; the uptake of labeled CO₂ and orotic acid was very low, but that of labeled deoxycytidine and thymidine was high.

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Arginase was found to be widely distributed in human urogenital tumors. Among the tissues examined, high enzyme activity was observed in benign prostatic hypertrophy and adenocarcinoma of the prostate, moderate activity in bladder tumors and Grawitz tumor of the kidney, and low activity in seminoma of the testis. Treatment of prostatic adenocarcinoma with estrogen decreased arginase activity. Ratio of activity in 12, 000g supernatant to that in 12, 000g precipitate from various urogenital tumors was in a similar value, but the ratio of papillary carcinoma of bladder differed from that of transitional carcinoma of the same organ. In the case of Grawitz tumor, arginase activity in tumor tissues was generally lower than that in the respective adjacent non-tumorous tissues of the kidney. Furthermore, the enzyme activity in adjacent non-tumorous tissues of multiple nodular type of Grawitz tumor was lower than that of a single nodular type, and was also lower when compared with the activity in kidney tissues of non-tumorous diseases.
Michaelis constant of arginase in various human urogenital tumors examined was almost similar in the value. Rate of activation of arginase from various urogenital tumors in the presence of Mn²⁺ was almost identical except benign prostatic hypertrophy, in which remarkable higher activation in the presence of Mn²⁺ was observed than that observed in other tumors.

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Dimethanesulfonylthiopentane (DSTP) inhibited the growth of Ehrlich solid carcinoma in mice when given repeatedly by intraperitoneal injection. The tumor cells which had been pretreated with DSTP before inoculation also failed to grow in the mouse groin. This finding suggests that the antitumor effect of DSTP may be produced by a direct action on tumor cells and not by a host-mediated action. The growth of Ehrlich ascites carcinoma was not affected by these treatments. The antitumor activity was not in parallel with the injury of the cell membrane by DS-TP. The enzymic activity of a hexokinase preparation from the tumor cells was inhibited by DSTP. The oxygen uptake of tumor cells depressed by DSTP was recovered completely by glucose and the tricarboxylic acid cycle intermediates, except citrate and isocitrate, and partially by glucose 6-phosphate and fructose 1, 6-diphosphate. A palmitate-albumin complex did not, however, restore the depressed oxygen uptake. A possible mechanism of the antitumor action of DSTP may be the inhibition of some enzymes involved in the glycolysis and fatty acid oxidation in tumor cells.

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Examinations were made on the interaction between some biologically active quinoline derivatives and calf thymus histone, and 13 of its constituent aliphatic amino acids. The interaction systems of histone and quinolines produced a difference spectrum in the visible region and intensity of this difference spectrum increased by heat denaturation of histone. The 13 aliphatic amino acids were classified into three kinds by the manner of their interaction with quinolines; glycine, alanine, leucine, isoleucine, valine, glutamic acid, and aspartic acid did not show any tendency to interact with the quinolines. Arginine, lysine, and proline, which have basic group with pKa larger than 10, produced a new absorption band in the visible spectral region which did not change with time. Methionine, serine, and threonine, which have a nucleophilic group in their molecule, produced a visible difference spectrum by interaction with the quinolines and the spectral intensity increased with time. The complexes formed between basic amino acids and 4-nitroquinoline 1-oxide were analyzed on the basis of Benesi-Hildebrand formula. It was concluded that an n-π charge transfer between quinolines and basic amino acid moiety of the macromolecule played an important role in the intermolecular interaction between the quinolines and histone.

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The effect of differential hypothermia, to keep tumor normothermic and general body hypothermic, on the cheek-pouch tumor of a Syrian hamster was investigated by an electron microscope.
In the tumor cells, condensation of nuclear chromatin, cystic dilatation of perinuclear cisternae, swelling of mitochondria, and disappearance of mitochondrial cristae were observed immediately after a 10-hr differential hypothermia. Vacuolization in the nucleus of some tumor cells was observed 12hr after the treatment, while the fibrocytes or fibroblasts in the submucosa of the cheek-pouch preserved their normal appearance almost wholly even 12hr after the treatment. On the other hand, aggregated intracisternal C-type virus particles of about 100nm in diameter were observed 12hr after the treatment.
Although the destruction of the nuclei of tumor cells became conspicuous, the membrane of some mitochondria still preserved its normal appearance even 24hr after the treatment.

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This experiment was conducted to study teratogenesis and carcinogenesis in relation to organogenesis, using the properties of short-action and high placental permeability of urethan.
Pregnant mice were exposed to urethan on various days of gestation (day 5 to 19) by a "single" injection, and malformations and neoplasms were induced in their offspring. Lung and tail anomalies were induced in 70-80% of the day 9 urethantreated group only, and this same group also developed a malignant teratoma. Pulmonary tumors were induced in high incidence in the groups exposed to urethan after day 13 of gestation. Embryological studies revealed that lung buds of mouse fetuses of this strain appeared on day 12. The same chronological relation was observed in induced hepatomas. Histologically, most of these pulmonary tumors were papillary adenomas. When the injection-parturition interval was short and late in term, pulmonary tumors were induced in higher incidence. Actual incidence of pulmonary tumors decreased in the day 19 urethan-treated group, when compared to that in the day 15 or 17 urethan-treated group. About one-third of mothers developed endometrial hyperplasia and hemangioma of uterine or placental origin.
When urethan was injected to lactating mothers, pulmonary tumors were induced in more than 50% of sucklings. This indicates that sucklings were exposed to urethan through the mother's milk.

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Immunohistological studies on rectal carcinoma, using the fluorescein isothiocyanate-labeled antibodies against gastric and intestinal mucosal glycoprotein, revealed that the less-differentiated or anaplastic carcinoma cells were well fluorescent while the well-differentiated ones of non-secretory activity were devoid of fluorescence. The less orderly the glandular structure was arranged, the more extensively and intensely the fluorescence developed. Such a relationship was recognized to exist in rectal carcinoma as well as in gastric carcinoma reported previously.
Mucosal glycoprotein in rectal carcinoma cells of secretory activity was found to possess immunochemical properties of both gastric and intestinal glycoprotein. In these cases, too, the profile of antigenic diversion was affirmed. Mucinous degeneration was more common and extensive in rectal carcinoma than in gastric carcinoma. Accumulated mucinous materials were intensely fluorescent with both of these antibodies.

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This study was made on four strains of Yoshida ascites hepatoma, AH-130, -41B, -57B, and -7974F, to examine the grades of differentiation and enzymic deviation from normal hepatocytes of an adult rat.
Ultrastructurally, these cells revealed various degrees of dedifferentiation from normal hepatocytes and partially transitional structures to bile duct type of cells.
The studies on acid phosphatase and non-specific esterase showed that these ascites hepatomas strongly deviated from hepatocytes. The isozyme pattern of acid phosphatase in ascites hepatomas was the fetal liver type characterized by the presecne of Band A and lacking in Band B of adult hepatocyte type, except that AH-41B appeared to be a more undifferentiated type. As for esterase isozyme pattern, these ascites hepatomas revealed Bands 2, 4, and 5, while they lacked Band 3 of adult hepatocyte type and were also defective in Bands 1 and 6 which are related to immature state of hepatocytes.
It was suggested that during successive transplantation (1) ascites hepatoma cells tend to lose the characteristics transmitted from the original hepatocytes and to reveal a non-hepatocytic feature, (2) these cells undergo various degrees of changes in the fine structure of cell organelles and in enzyme properties, independent of one another, and ultimately (3) they reach a primitive type of cells.

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In order to study the characteristics of Yoshida sarcoma, the cell membrane of which was modified with a SH-blocking agent, 6, 6'-dithiodinicotinic acid, kinetic reaction of the tumor cell was examined with the ³H-labeled compound. This reaction proceeded very rapidly with formation of mixed disulfides between membrane SH groups and half moiety of this compound, and with a gradual release of 5-carboxy-2-thiopyridone into the medium, reaching a saturated value by incubation for 10min at 37°. Thirty percent of all the mixed disulfides of the modified cell were found to have labile disulfide linkage. From an aspect of cancer chemotherapy, the results so far obtained indicate that the modified cells had no effect on sensitivity to nitrogen mustard, and the combination of this compound and nitrogen mustard showed only an additive effect.

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Cases of liver cirrhosis and primary hepatic cancer were studied from autopsy cases at the Department of Pathology, University of Cambridge, England, and the result was compared with the one of the same kind obtained from the Department of Pathology of this University. The main points of note are as follows:
(1) Total incidence of non-specific (portal and postnecrotic, or postnecrotic, posthepatitic and nutritional) liver cirrhosis among autopsy cases is higher in Tokyo than in Cambridge, while that of specific (e.g., biliary, cardiac, etc.) cirrhosis does not differ much between the two cities.
(2) Total incidence of primary hepatic cancer is remarkably higher in Tokyo.
(3) There is an agreement in the fact that significant causative relation seems to exist only between hepatocellular carcinoma and non-specific cirrhosis.
(4) Among non-specific cirrhosis, types A (postnecrotic) and B (posthepatitic) are exclusively related to the occurrence of hepatocellular carcinoma (hepatomarelated cirrhosis), and type C (nutritional) is not (hepatoma-unrelated cirrhosis).
(5) Hepatoma-related cirrhosis is not only more common in number, but also has a more possibility of associating with primary hepatic cancer, in Tokyo. Hepatomaunrelated, non-specific cirrhosis is much more common in Cambridge.
(6) Cholangiocellular carcinoma seems to have no cause-and-effect relation at all to any type of liver cirrhosis of non-specific group.
(7) Specific type of liver cirrhosis seems to have no relation to the occurrence of any primary hepatic cancer.
(8) As a conclusion, the geographic-pathological status of liver cirrhosis and primary hepatic cancer in Cambridge, U.K., is rather similar to the one of Cincinnati, Ohio, U.S.A., and much different from the one of Tokyo, Japan. On the other hand, as a matter in general, a possible role of viral hepatitis as a cause of hepatocellular carcinoma is again suggested.

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A technique is described for the production of colonies of suspension-grown L-1210 mouse leukemia cells (L-1210/C), L-5178Y mouse lymphoma cells (L-5178/C), and Yoshida ascites sarcoma cells (YAS/C) in 11 days in a medium, RPMI-1640, containing 20% calf serum and 0.4% Bacto-agar, and incubated at 37° in a CO₂ chamber. With the development of the single cell culture technique, with a colonyforming efficiency of 99.5% in L-1210/C, 74.5% in L-5178/C, and 28.0% in YAS/C, it became possible to apply it to studies on cytocidal action of compounds for use in cancer chemotherapy.
The effect of Mitomycin-C has been quantitatively studied on single cells of L-1210/C and YAS/C. Survival (S) of the single cells (defined as the ability to form macroscopic colony within 11 days) yields a linear logarithmic line when plotted against Mitomycin-C concentration (D). Survival rate fits the equation log S=log n-kD. Reasons for this linea rlogarithmic survival line by Mitomycin-C are discussed.
The sensitivity of cell to Mitomycin-C can be indicated by the mean lethal dose (MLD); a reciprocal of slope coefficient k, which shows the concentration of Mitomycin-C needed to reduce survivors to 10% in use of common logarithm, or 37% in use of natural logarithm. On this basis, the sensitivity of YAS/C cells to Mitomycin-C is about 100 times greater than that of L-1210/C cells. The value of MLD becomes smaller with a longer exposure to Mitomycin-C and the value at 11-day exposure is about one-hundredth of that of 30-min exposure in both cell lines.

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In the culture of several mammalian cell lines (HeLa S3, rat embryonic fibroblast, Swiss 3T3, rat lung carcinoma cells, and KB), specific biological and biochemical alterations were induced when the culture was carried out with no medium change for 10-18 days in the presence of a specific sugar or an amino-sugar related to cell membrane in a concentration ranging from 3 to 30mM.
The specific alterations induced were (1) higher number of substrate-attached cells (survival cell number) in prolonged cultivation, (2) higher total number of cells per flask, and (3) greater viscosity and stronger positivity in mucin clot test of the medium, corresponding to a greater amount of hyaluronic acid accumulated.
Among the membrane-related sugars, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetyl-D-mannosamine, bis(N-acetyl)chitobiose, tris(N-acetyl)chitotriose, α-methyl-D-glucopyranoside, and D-glucose were effective in inducing the above alterations. D-Glucosamine, L-fucose, L-glucose, S-methyl-D-glucoside, and N-acetylneuraminic acid were ineffective.
It was demonstrated electrophoretically that HeLa S3 cells synthesized hyaluronic acid in culture and the synthesis was much enhanced by the presence of N-acetyl-D-glucosamine in the medium.

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A study was conducted on the adhesion of Ehrlich ascites tumor cells to the surface of homologous mouse peritoneal macrophages. The tumor cells, upon treatment with concanavalin-A, a plant agglutinin, displayed a remarkable adhesion to macrophage cell surface. Tumor cell adhesion took place also when macrophages, instead of tumor cells, were pretreated with concanavalin-A. The concanavalin A-induced adhesion of tumor cells was inhibited by D-glucose or α-methyl-D-glucoside, specific inhibitors of concanavalin-A action. Similar phenomena were observed with concanavalin A-treated Sephadex granules. They adhered to macrophages, and agglutinated with themselves or with tumor cells, and these activities were inhibited by the same sugars.
These results suggest that a common mechanism might exist between tumor cell adhesion to macrophages and tumor cell agglutination induced by concanavalin-A, and this is probably due to the formation of agglutinin bridges between the two cell types.

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Viral RNA in the cytoplasm appears as a sharp single peak (19-20S) in the rat embryo cells transformed by adenovirus type-2. The remarkable difference from other data is the lack of larger RNA component (over 25S).

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This communication reports studies on hepatocarcinogenic effects of polychlorinated biphenyls (PCB) in dd mice. The hepatomas induced in mice by Kanecrol-500 of PCB appeared similar to those induced by the α-isomer of benzene hexachloride, but Kanecrol-400 and Kanecrol-300 of PCB had no carcinogenic activity in the liver of mice.

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