Two epithelial cell lines (TE-1 and TE-2) have been established from a well or poorly differentiated human squamous cell carcinoma of the esophagus. TE-1 has been subcultured 120 times during 2 years and 10 months, and TE-2, 50 times for almost 2 years. Cultured cells grew as isolated and piled-up colonies of epithelial cells. The average doubling time of the TE-1 cell line was 60hr and that of TE-2, 72hr, Distinctive marker chromosomes and a male karyotype were present in TE-1, but no marker chromosomes were seen in TE-2. Scanning electron microscopic examinations of both TE-1 and TE-2 confirmed the presence of desmosomes and interdigitated microvilli. Transmission electron micrographs of TE-1 showed the presence of abundant cell organelles, and a few organelles were found in the scanty cytoplasm of TE-2. There was a marked difference in the cell organelles between TE-1 and TE-2. Heterotransplantation of the cultured TE-1 and TE-2 cells produced tumors, the histological appearance of which was similar to that of the original ones. The carcinoembryonic antigen level of the medium in the confluent culture of TE-2 was 270ng/10⁶ cells.
In the cytoplasm of TE-1 cells the number of paracrystals, which were produced by treatment with vinblastine sulfate, increased by the addition of cholera toxin to the medium.

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The toxicity of lectins from castor bean (Ricinus communis L.), ricin-D, ricin-E, and castor bean hemagglutinin, was investigated on five cultured cell lines. The differential effect of their constituent polypeptide chains was also investigated using these cell lines. Ricin-D, ricin-E, and castor bean hemagglutinin (CBH) possessed cytoagglutinating activity and cytotoxic activity to all five cell lines. These lectins showed the strongest toxicity to L5178Y cells, which are leukemic cells. The toxic activity of ricin-D was stronger than that of CBH in all cell lines. The constituent polypeptide chains of ricin-D and CBH were separated by DEAE-cellulose chromatography and designated as isoleucine chain and alanine chain denoted by their N-terminal amino acids. Only alanine chain of ricin-D was toxic to cells grown in vitro, whereas isoleucine chain of ricin-D and alanine chain of CBH were not toxic to the cells.
Moreover, it was found that both lectins caused syncytium formation in NIH3T3 cells infected with Moloney leukemia virus and this cell fusion activity was shown to be exclusively associated with the alanine chain. Cytotoxic, cell agglutinating, and syncytium forming effect of the lectins is due to binding of the alanine chain of ricin-D to galactose-like residues of the membrane constituents of these cells.

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Iron uptake rate of the tumor tissues of the rat liver was compared with that of the nontumor tissues of the same rat liver. Ferric [⁵⁹Fe] citrate was injected to rats bearing hepatoma induced by the administration of diethylnitrosamine. The tumor and nontumor tissues were enucleated from the liver and radioactivity (cpm/10mg tissue) of the individual tissue was calculated to estimate the iron uptake rate of the tissue. Eleven rats were finally available for the study.
Population mean of radioactivity of the tumor group was larger than that of the nontumor group when examined 6hr after the injection, so far as the specimens of both groups were sufficient in number, more than 19. The variance of radioactivity of the tumor group was always larger than that of the nontumor group, probably in part due to cytological and histological varieties of the tumor tissues.
These results suggest that the tumor tissues can take up more iron than the nontumor tissues.

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A highly lymphotropic derivative of a proteinaceous antitumor agent, Neocarzinostatin, was prepared by chemical conjugation of a water-soluble synthetic poly(maleic acid)-styrene oligomer. The derivative of 2.5 × 10⁴ dalton exhibited a strong antitumor activity against AH109A and DBLA-6 as well as antimetastatic activity against metastatic AH109A with which experimental lymphatic metastasis was produced in rats. An increased lipophilicity and molecular weight of the derivative appear to be responsible for its improvement as lymphotropic antimetastatic agent.

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Induction in vivo of resistance of P388 mouse leukemia to typical antitumor agents was studied. Rapid acquisition of resistance was observed with adriamycin, vincristine, β-D-arabinofuranosylcytosine, and 6-mercaptopurine. Complete resistance to these agents was observed at 2nd transplant generation. Sensitivity of the cells to actinomycin-D, 5-fluorouracil, and nitrogen mustard decreased stepwise and steadily, resulting in complete resistance at the 5th to 10th transplant generation. In the case of methotrexate, sensitivity to this agent was significantly reduced even at the 2nd generation, and the tumor acquired complete resistance at the 5th generation. It was hardly possible to induce resistance to mitomycin-C by a similar procedure.
Cellular resistance to these antitumor agents remained unchanged in the absence of selection pressure over 10 transplant generations in spite of the fact that long-term drug treatment was discontinued at the 2nd generation after a complete resistance was induced.
Degree of resistance at the cellular level was observed with each subline by in vitro culture technique and compared with each other. The highest resistance was acquired to actinomycin-D and β-D-arabinofuranosylcytosine and the degree of resistance to adriamycin, vincristine, and nitrogen mustard was moderate. Although P388 sublines, resistant to 5-fluorouracil and methotrexate, did not show any response in vivo to the corresponding drug, degree of cellular resistance was found to be relatively low.

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Crude tumor cell extract from rat ascites hepatoma AH130 revealed a high thromboplastic activity. Intravenous injection of the extract caused widespread thrombus formation in the capillaries, arterioles, and arteries of the lung. Intravenous inoculation of AH130 after an injection of the tumor cell extract produced metastatic foci in the larger arteries, compared with the case injected with only AH130 which developed metastatic foci mainly in and around the alveolar septa. These results suggest the role of tumor thromboplastin material in the mode of distribution of metastatic foci in the lung.

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Of 62 cases of histologically confirmed osteosarcomas, 50 long bone cases aged under 30 years were grouped as "typical" osteosarcoma, and the other 4 long bone cases and 8 short and flat bone cases were grouped as "atypical" osteosarcoma. Gross and microscopic findings of the lesions, postmortem findings, and survival of the patients were compared between the two groups.
Small round cell-type tumor cells and relatively well-differentiated foci resembling osteoblastoma were more often found in the atypical than in the typical osteosarcomas. Although patterns of metastasis were not very different between the two groups, 2 of 6 autopsied short and flat bone cases showed no metastasis. Five-year survival rates of both groups were similar, each about 25%. Six patients of the typical and only one of the atypical osteosarcomas are alive without recurrence more than 6 years after amputation.

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The effect of phenobarbital on simultaneous induction of liver and lung tumors was examined in inbred DDD mice. Group 1 of newborn mice received a single intraperitoneal (ip) injection of dimethylnitrosamine (DMN) and after weaning they were given 0.05% phenobarbital solution to drink. Group 2 received an injection of DMN like Group 1 but were then given normal water. Group 3 were injected ip with 0.9%NaCl solution and then given phenobarbital solution to drink as in Group 1, and Group 4 were injected with 0.9%NaCl solution like Group 3, and then given tap water to drink. The animals were examined 16 weeks after birth. In Group 1, 27 of 35 mice (77%) had liver tumor and 15 (43%) had lung tumor. In Group 2, 8 of 24 mice (33%) had liver tumor and 16 (67%) had lung tumor. Animals in Groups 3 and 4 did not develop tumors. The difference in the incidences of liver tumor, but not lung tumor, in Groups 1 and 2 was statistically significant (P<0.01); that is, a promoting effect of phenobarbital was observed in induction of liver tumor, but not lung tumor.

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The effect of 2-naphthylamine and cyclophosphamide on the urinary bladder and liver of female BALB/c mice was investigated. The bladder mucosa of mice treated with 2-naphthylamine alone for 40 weeks showed diffuse hyperplasia. Oral administration of 2-naphthylamine for 40 weeks plus injections of cyclophosphamide produced bladder carcinomas in 30.8∼35.7% of the animals, associated with downward growth of the bladder epithelium. All the bladder carcinomas were of the transitional cell type and most of them contained pseudoglandular areas. Hepatomas seemed to develop in higher incidence in mice treated with 2-naphthylamine plus cyclophosphamide than in mice treated with 2-naphthylamine alone. Most of the hepatomas were solitary and showed a trabecular pattern. Cyclophosphamide seemed to have a summative or promoting effect on carcinogenesis of the bladder mucosa and liver induced by 2-naphthylamine in female BALB/c mice.

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Carcinogenic effect of N-ethyl-N-nitrosourethan (ENUR) and N-amyl-N-nitrosourethan (ANUR) was examined by continuous oral administration or topical application to female Donryu rats. Oral administration of 100ppm solution of ENUR induced 100% of tumors in the forestomach, 46%, 80%, 71%, and 51% in the oral cavity and pharynx, esophagus, duodenum, and liver, respectively. On the other hand, the incidence of forestomach tumors was 78%, that of oral cavity and pharynx, and esophagus was 93% and 98%, respectively, in rats given 400ppm suspension of ANUR.
In addition, topical application of ENUR induced tumors of the skin as well as tumors of the forestomach and liver.

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Mutagenic and DNA-damaging effects of a series of N, N-dialkylnitrosamines monosubstituted at the α-carbon with an acetoxyl group were tested in Salmonella typhimurium, Escherichia coli, and Bacillus subtilis in the absence of metabolic activation system. The compounds comprised 8 N-alkyl-N-(acetoxymethyl)nitrosamines (alkyl=methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, or tert-butyl) and N-butyl-N-(1-acetoxybutyl)nitrosamnie. All the compounds, except one with a tert-butyl group, gave positive results in these mutagenicity and repair tests. Presumed release of alkyl cations from the corresponding α-acetoxy derivatives by hydrolysis and heterolysis caused mutagenic and DNA-damaging effects in the bacteria. Structure-activity correlation of the compounds was noted in these tests and discussed in regard to the mutagenicity with metabolic activation and carcinogenicity of N, N-dialkylnitrosamines. The results support the hypothesis that α-carbon hydroxylation is one probable mechanism involved in the metabolic activation of N, N-dialkylnitrosamines.

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A soluble factor which suppresses phagocytic ability of macrophages against Staphylococcus aureus, as well as latex particles, was found in the culture supernatant of sarcoma-180 cells. This factor was stable to heating at 56° for 30min and had a molecular weight of more than 10, 000. Supernatant from normal spleen cell culture also showed some antiphagocytic activity but it was considerably lower than that of sarcoma-180 cell culture.

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Tissues from the surgical specimen of 7 patients with testicular embryonal carcinoma were cultivated in vitro. Among 7 primary cultures, initial growth of tumor cells was observed in 5 cases. One of them was successfully subcultured and a cell line, designated NEC-8, has been established. NEC-8 cells grow as a fiat epithelial colony densely packed with polygonal cells and gradually pile up at the center of the colony. Ultrastructure of NEC-8 cell revealed desmosomes and microvilli at the cell membrane. The cytoplasm was characteristically rich in glycogen granules which corresponded with strong PAS reaction. The chromosome number of NEC-8 cells at passage 3 showed wide distribution from 70 to 183, with a mode of 85 to 88. Histology of the heterotransplant of NEC-8 cells into the hamster cheek pouch was that of adenocarcinoma resembling a part of the histology of the original tumor.

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The effect of hyperthermia (43∼45°) on DNA single-strand breaks induced by bleomycin was examined in HeLa cells by the alkaline sucrose gradient sedimentation analysis. When HeLa cells were exposed to a temperature of 43°∼45° for 1hr, only a small number of DNA single-strand breaks were produced. Treatment with bleomycin at 43° or 45° induced more DNA single-strand breaks than bleomycin treatment at 37°. The DNA strand breaks induced by bleomycin were almost completely repaired by post-treatment incubation of the cells in the control medium at 37°. On the contrary, the repairing capacity of the cells was markedly inhibited by post-treatment incubation at 43°, and this inhibition of cell repair seemed irreversible because no repair was found after an additional incubation of the cells at 37° for 1hr.
These results support the possibility that the mechanism of synergism between the effect of bleomycin and hyperthermia may be related to the inhibition of DNA repair.

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The effect of chemotherapy combined with immunostimulants on the activities of macrophages in mice was studied. The number of macrophages and exudate cells in the peritoneal cavity increased 3 days after ip injection with mitomycin-C, cyclophosphamide, and 5-fluorouracil together with OK-432 or yeast cell wall and decreased to normal level after 9 days, while the number of the cells remained decreased in mice receiving multi-drugs alone. Acid phosphatase activity of the macrophages of mice was elevated after the simultaneous injection of yeast cell wall and OK-432, and high activity was preserved in the macrophages of mice receiving yeast cell wall even after 9 days. Spreading of these cells was also enhanced. Macrophage activities examined by these assays were maximal in every respect 6 days after combination therapy. Cytostatic activity of the cells was strengthened after 6 days by combined use of OK-432 or yeast cell wall. Role of the activated macrophages in combination therapy was discussed.

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The binding of radioactivity from 1, 1'-ethylene-bis(1-nitrosourea) (EBNU), which has antitumor activity, 1-methyl-1-nitrosourea (MNU), 1-ethyl-1-nitrosourea (ENU), and 1-propyl-1-nitrosourea (PNU), labeled with ¹⁴C in either carbonyl or alkyl group, to nucleic acids, proteins, and synthetic biopolymers was compared in vitro. The binding of radioactivity from ¹⁴C-carbonyl group of EBNU to proteins and polypeptides was at least two-fold higher than that of MNU, ENU, and PNU. On the other hand, each N-nitrosourea has some different specificity for binding of radioactivity from ¹⁴C-alkyl group to nucleic acids, proteins, and synthetic biopolymers. The alkylating activity to DNA decreased in the order of MNU, EBNU, and ENU. PNU did not bind with DNA to any detectable extent. The binding of radioactivity from EBNU[ethylene-¹⁴C] was found in histone, but that from each mono-N-nitrosourea-[alkyl-¹⁴C] was not found. The effect of various amino acids on the binding of ¹⁴C-EBNU and ¹⁴C-MNU to polylysine and poly (G) was studied. All the amino acids tested had no or little effect except cysteine. The binding of radioactivity from carbonyl-¹⁴C group of EBNU and MNU to polylysine decreased about 30% and 20%, respectively, in the presence of equimolar amount of cysteine. Cysteine completely inhibited the binding of radioactivity from EBNU[ethylene-¹⁴C] to polylysine and poly (G), but a little from MNU[methyl-¹⁴C] to poly (G).

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The effect of chemo-immunotherapy on lymphocyte responsiveness to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) was investigated in patients with advanced cancer. Patients received OK-432, a streptococcal preparation, intratumorally and additional systemic MFC (mitomycin-C, 5-fluorouracil, and cytosine arabinoside) chemotherapy intravenously. Patients with high lymphocyte responsiveness to mitogens at the beginning of treatment were more responsive to chemo-immunotherapy than those with low responsiveness. Furthermore, lymphocyte responsiveness to PHA was maintained at a high level throughout the treatment. In contrast, patients with initially depressed lymphocyte responsiveness maintained low responsiveness throughout the treatment. A significant increase of inhibitory activity of the serum was observed in patients with poor results of treatment. From these results, it seems reasonable to conclude that the response to chemo-immunotherapy might be predicted by in-vitro lymphocyte responsiveness.

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Under simulated human gastric conditions, glycocyamine which exsists in meat is known to be converted into N-carboxymethyl-N-nitrosourea (CMNU) by reaction with sodium nitrite. Because of its suspected hazards to man, CMNU was tested for its mutagenicity and lethal activity with a set of isogenic strains of Escherichia coli possessing the same auxotrophic marker but different DNA^- repair capacities. Both strains NG30 (recA^-) and R15 (polA^-) were far more sensitive to lethality induced by CMNU than H/r30R (wild) and Hs30R (uvrA^-) strains. The uvrA^- strain was more sensitive to induction of mutations by CMNU than the wild and polA^- strains, but the recA^- strain was hardly mutable by CMNU. It can be concluded from these findings that the major cause of lethality of CMNU in E. coli is different from that of mutation induction.

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Examinations were made to see if various vitamin A compounds, including retinol, retinal, retinol palmitate, retinol acetate, and retinoic acid, enhance the action of fusidic acid, an antibiotic and inhibitor of protein synthesis, on human carcinoma HeLa cells in vitro. Among these vitamins, retinal and retinol were found to potentiate strongly the effect of fusidic acid on HeLa cells. In this system, however, retinol palmitate, retinoic acid, and retinol acetate in 10∼20μg/ml concentration were not found to enhance the effect of fusidic acid significantly.

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Stage IIIa breast cancer patients with a high titer of sex hormone-binding globulin (SHBG) had a disease-free interval of 19 months (±5 months standard deviation) compared with 10±6 months for patients with low SHBG levels. The difference was significant (P<0.01).

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Fractional distribution of 51 Cr-erythrocytes, 125 I-human serum albumin, and 86 RbCl was used to determine, respectively, vascular volume, permeability, and perfusion in rat urinary bladder during carcinogenesis by N-butyl-N-(4-hydroxybutyl) nitrosamine.

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Types I and III vascular foci were found in carcinogenesis by 7, 12-dimethylbenz[a]anthracene (DMBA). Type I foci may represent repair of direct damage to blood vessels by DMBA. Type III I foci showed close relationship to epithelial proliferative lesions, i.e., papillary or nodular hyperplaisa, papillomas, or carcinomas.

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