The present study was designed to examine the epidemiological association of food consumption with cancer mortality in Japan, if any, in comparison with similar examinations in developed countries including Japan, in a search for dietary factors in cancer etiology. Whereas food consumption patterns in Japan was believed to be specific among developed countries, this study revealed that the correlation matrix among major foods both for international and Japanese data made it possible to categorize these foods into two groups with some similar patterns, since there should be observed a positive or negative correlation for each pair of foods. The patterns for correlation of food consumption to cancer mortality in Japan were found to be considerably different from those in developed countries, and this suggested that the epidemiological association between the two in Japan, if any, could be specifically evaluated from that in developed countries. The urban-type region for food consumption in Japan, however, indicated a possible westernization for the relation of food consumption to cancer mortality as well as for food consumption patterns, whereas the traditional Japanese region for food consumption did not indicate any similarity with westernization for the relationship between food consumption and cancer mortality, as well as for food consumption patterns. The above findings also suggested that there might be a possible significance for the regionalization of Japan in such studies, and that the urbanization of food consumption patterns in Japan would show that this country has a similar pattern of association between food consumption and cancer mortality as observed among developed countries in Europe and/or North America.

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Mechanism of natural resistance of rat ascites hepatomas to nitrogen mustard was examined with tumors, especially AH-13 and AH-44, sensitive and resistant to the chemical, respectively. There was found little difference in the uptake and binding of the chemical among the two lines. In contrast, DNA repair activity of the resistant line was higher than that of the sensitive line. This result suggested that natural resistance of AH-44 to the drug may be, at least partially, due to its higher activity of repairing DNA damage.

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In culture, a cloned rat liver cell line, J-5-2, exhibited a high diploidy for 200 days after the last cloning (diploid line) but, thereafter, pseudodiploid cells gradually increased in number (pseudodiploid line). These diploid and pseudodiploid lines were inoculated at various sizes, and chromosome analysis was performed for 75 days of serial passages. Diploid line showed an increase of pseudodiploid cells at a large inoculum size. On the other hand, more than 80% of the cells examined maintained the diploid karyotype at a small inoculum size. Pseudodiploid line showed an increase of pseudodiploid cells at a large inoculum size and of diploid cells at a small inoculum size. These pseudodiploid cells contained three types of marker chromosomes, in which #1 chromosome was found to be involved by the G-banding methods.
The saturation densiy of the pseudodiploid line was much higher than that of the diploid line, and plating efficiency of the diploid line was also significantly higher than that of the pseudodiploid line, although the population doubling time of these two lines was almost the same.
Possible mechanisms for the effect of different inoculum sizes on the incidence of diploid cells and pseudodiploid cells are discussed.

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A surgical specimen of solitary, encapsulated tumor tissue obtained from a 52-year-old male, diagnosed histologically as well-differentiated hepatocellular carcinoma (Grade II, Edmondson and Steiner) with liver cirrhosis, Type A' (and B in some parts), was found to have a supernormal level of pyruvate kinase Type L and subnormal level of Type M₂; the activities (units/mg protein) being 1.21 and 0.12, respectively. The resulting isozyme pattern was apparently "superdifferentiated" as compared with those of not only the tumor-bearing, cirrhotic liver (Type L, 0.19; Type M₂, 0.67) but also the normal liver (Type L, 0.47±0.05; Type M₂, 0.18±0.02). The electrophoretic and kinetic properties of the type L isozyme were identical with those of the cirrhotic host liver and a non-cirrhotic control liver. Other enzyme levels in the hepatoma tissue were as follows: Glucose-6-phosphatase, normal; fructose-1, 6-bisphosphatase, reduced; glucokinase, absent; and hexokinase Types I and III, and glucose-6-phosphate dehydrogenase, slightly increased. The serum α-fetoprotein level was 95ng/ml. The whole enzyme profile is consistent with the minimal deviation hepatomas in rats. The results were compared with those of other human hepatomas, and the mechanisms of disordered regulation in hepatoma gene expression were discussed.

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Three cell lines were established from embryos of DDD, AKR, and C3H mouse strains based on "3T3" transfer schedule. These cell lines were designated as Y-DD, Y-AK, and Y-CH. Y-DD and Y-CH showed a gradually increasing growth rate during the course of their establishment, whereas Y-AK indicated a constant growth rate, a low level of saturation density, and a tetraploid range of chromosome mode. In transformation experiment, control cultures of Y-DD and Y-CH already lost a density-dependent inhibition, but Y-AK was found to be very sensitive to density-dependent inhibition. Y-AK showed a higher activity in benzo [a] pyrene metabolism (4100pmol/10⁶ cells/24hr) than Y-DD and Y-CH (700 and 2600pmol/10⁶ cells/24hr).

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In a syngeneic mouse-tumor system, anti-thymocyte serum treatment nullified tumor suppression mediated by BCG (living and nonliving) and living C. liquefaciens, but not the effect of nonliving C. liquefaciens. Treatment with anti-macrophage serum or latex particles also impaired the efficacy of living BCG but had no effect on that of C. liquefaciens.

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Peripheral blood lymphocytes from patients with uterine cervical cancer were tested against a cervical cancer cell line by micro-cytotoxicity assay. In stage 0, high degree of cytotoxicity was observed but it decreased with progression of disease. In stage IV and recurrent cases, cytotoxic activity was negligible. Lymphocytes from healthy controls and myoma patients were not cytotoxic against target cells. On the other hand, lymphocyte response to phytohemagglutinin as a nonspecific T-cell activity was as high as that of healthy control in stage 0, but the response decreased in stages I to IV and recurrent cases. In stages I to III, several cases showed cytotoxic activity but decreased response to phytohemagglutinin.

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The glycosaminoglycans were prepared by exhaustive Pronase digestion and alkaline treatment of squamous cell carcinoma and adenocarcinoma tissues of human lung, and of tissues taken at a site distant from the tumor as a control.
The glycosaminoglycan classes were characterized by chemical, enzymic, and electrophoretic methods. The presence of oversulfated chondroitin- and/or dermatan-sulfates which have not up till now been found in lung tissues was also demonstrated in carcinoma and control tissues, their contents being higher in the carcinoma tissues.
The levels of whole glycosaminoglycans were markedly increased in carcinoma tissue. The classes of glycosaminoglycans which increased in lung carcinoma tissue were predominantly chondroitin-4- and/or -6-sulfates and hyaluronic acid.

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Quantitative study was carried out on transcerebral passage of tumor cells using Yoshida sarcoma and 6 strains of rat ascites hepatoma. The counting was done every second for 40sec. In relation to the results obtained, histological study was made on the cerebral metastasis of these tumor cells. Metastatic foci in the brain parenchyma, except the meninges and choroid plexus, were formed by island-forming strains (AH-130, AH-272, and AH-7974) which showed comparatively low passage rate and not by single-cell strains (Yoshida sarcoma, AH-7974F, AH-66F, and AH-13) which showed comparatively high rates. Accordingly, it is obvious that there is a correlation between transcerebral passage rates and brain metastasis formation. The mechanisms of metastasis formation were discussed with special reference to arrest of tumor cells in blood vessels.

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Growth behavior in vitro of 3T3 cells and the cells transformed by polyoma or SV40 virus was studied after their treatment with dextran sulfate. The transformed cells treated with dextran sulfate showed decreased saturation density compared with untreated cells, whereas saturation density of 3T3 cells was hardly affected by treatment with dextran sulfate. The ratio of saturation density of untreated SV3T3 cells to that of the treated cells was 64.1% at 5μg/ml and was 43.4% at 10μg/ml of dextran sulfate. In Py3T3 cells the ratio was 45.0% at both concentrations of 10 and 20μg/ml. Plating efficiency was not affected in 3 cell lines tested after treatment with dextran sulfate. The treated cells showed a tendency to form thin and not piled-up colonies in the central area. The treated SV3T3 and Py3T3 cells were flattened in shape compared with the untreated cells. In agar medium the treated SV3T3 and Py3T3 cells showed the tendency to stop growing after forming small colonies, whereas the untreated cells kept growing and formed large colonies. These results suggested that dextran sulfate altered temporarily the growth of the transformed cells to that of untransformed cells.

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Solutions of 100mg/kg 1-butylurea and 50mg/kg sodium nitrite were administered daily by a stomach tube to pregnant ACI/N rats from the 13th to 21st day of gestation. Neurogenous tumors were induced in their offspring. The incidence of tumors and mean survival time of rats with nervous tumors were 64% (23/36) and 309 (189∼672) days, respectively. Localization and histological findings of the nervous tumors were similar to those observed previously in rats whose mothers received 1-butyl-1-nitrosourea (BNU) during pregnancy.
Neurogenous tumors did not develop in the offspring of the mothers that received 100mg/kg of 1-butylurea alone in the same manner. In several rats of both groups, a few spontaneous tumors of the testis, pituitary gland, urinary bladder, uterus, and colon were detected.
In vivo formation of BNU in stomach content after intubation of 1-butylurea and sodium nitrite was determined. BNU was detected in the stomach content at levels of 25ppm at 30min and 23ppm at 60min after administration. The concentration of BNU in the stomach content corresponded to 48.3 and 29.2μg/rat, respectively.

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The previously observed alterations in the energy transducing system of rat liver mitochondria during 3'-methyl-4- (dimethylamino) azobenzene (3'-Me-DAB) carcinogenesis were investigated using aliphatic dicarbonyl compounds as molecular probes and the effect of temperature on the membrane-linked NADH-indophenol reductase. The vicinal diketone, diacetyl, uncouples oxidative phosphorylation in normal rat liver mitochondria while the higher diketones, acetylacetone and acetonylacetone, are increasingly less effective in that order; diacetyl totally abolishes respiratory control with substrates the oxidation of which involves the NADH→CoQ segment, but only partially with succinate which bypasses this segment. Diacetyl, likewise, uncouples oxidative phosphorylation in liver mitochondria from rats fed 3'-Me-DAB, but the mitochondria are most resistant to this uncoupling (in terms of the P/O ratio) at the time period when the respiratory control index (determined in the absence of diacetyl) is at the dye-induced minimum. This time period is at 3 to 4 weeks of dye administration, representing the cumulative dose for tumorigenesis threshold. At this threshold period of feeding 3'-Me-DAB, discontinuities in the Arrhenius plot of the mitochondrial membrane-localized NADH-indophenol reductase appear, with a return toward the control state (no break) at 8 weeks, only to reappear in the plot of the enzyme from tumor mitochondria, suggesting sequential membrane phase transitions in the mitochondria during azo dye carcinogenesis.

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Type-C virus particles were revealed by electron microscope in 6 of 9 tumors of cultured and biopsied human cancers heterotransplanted into nude mice. Some tumors in nude mice were explanted for in vitro cultivation. The virus particles were also found in the cultures derived from the virus-positive tumors. They were mostly found extracellularly, but the particles in budding process were also encountered frequently. Cytological study and karyotype analysis of the cultured cells proved these virus-releasing cells as of human origin.
From the close correlation between the statistical virus counts and the complement fixation titers for murine gs antigen of the tumors and their cultures, these viruses propagated in human cancer cells were confirmed to be infectious viruses of nude mouse origin.
The virus replicating in human cancer cells was readily infected in some of innocent human cancer cells by co-cultivation. It is to be emphasized that infection of animal endogenous viruses on heterotransplanted human cancer cells is a bothersome contamination for human cancer research, especially when searching for a human tumor virus candidate.

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³²P-Labeled adenovirus-12 (Ad-12) DNA was treated with restriction enzyme (EndoR•Hin dIII) isolated from Haemophilus influenzae (Rd strain) and the resulting 16 specific fragments were separated through gel electrophoresis. The kinetics of renaturation of each of the fragments was measured in the presence of unlabeled Ad-12-transformed hamster embryo cell, clone 9 (Ad-12HE-C19). More than 77% of the viral genome nucleotide sequence was present in Ad-12HE-C19 cell DNA with 5 to 10 copies per haploid quantity of cell DNA of each of the sequences of 11 fragments examined; A, C, D, E, F, G, H, I, J (J1+J2), K, and L. However, it is suggested that only a part of the nucleotide sequence in B fragment may be present in the cell DNA.

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The combined effect of anticancer agents (Mitomycin-C, cyclophosphamide, or 5-fluorouracil) and anaerobic Corynebacterium liquefaciens on subcutaneously induced solid Ehrlich carcinoma in mice was examined. Mitomycin-C and cyclophosphamide were given intraperitoneally on day 7 after inoculation of tumor cells. 5-Fluorouracil was administered intraperitoneally for 7 consecutive days from day 9 to 15. C. liquefaciens was given in two ways, intraperitoneally and intratumorally. Its injections were made on days (-7, -5), (-4, -2) (+2, +4), or (+5, +7) in the intraperitoneal groups and in every way varying from (+9, +10) to (+19, +20) days in the intratumoral groups. The best result was observed in combination of C. liquefaciens and 5-fluorouracil in the intraperitoneal groups and that of C. liquefaciens and cyclophosphamide in the intratumoral groups.
Although the results were not necessarily good, probably due to the poor design on time schedule of C. liquefaciens, they partly confirm the hypothesis that the activity of conventional anticancer drugs can be potentiated by a non-specific immunostimulation by anaerobic C. liquefaciens in the solid tumor of mice same as in the ascitic tumor reported previously.

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Five transplantable tumors, which originated from human maligant neoplasms and had been maintained in nude mice, were frozen at -70° and preserved for 12∼26 days. Tumor tissue was minced in a mixture of tissue culture medium, 10% dimethyl sulfoxide, and 10% calf serum before freezing.
The transplantability of frozen tumors was examined after thawing. Morphological and functional characteristics of each tumor were compared before and after the frozen storage. The results suggest that low-temperature preservation technique can be applied satisfactorily for the maintenance of human malignant tumors which have been maintained by using nude mice.

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6-Oxybenzo[a]pyrene radical formed by oxidation of 6-hydroxybenzo[a]pyrene with chloranil was found to bind covalently to DNA or polynucleotides. The bound complex was stable and binding ratio to poly(G) was far larger than those to other polynucleotides or DNA, indicating that guanine residue is the site of binding.

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Neovascularization during urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxy-butyl)nitrosamine was studied by scanning electron microscopic examination of vascular casts and by autoradiography with three distinct patterns discernible. The patterns were different in the early hyperplastic changes from those observed in carcinomas.

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