Cotransfection of a transformation-deficient and immortalization-positive SV40 early gene mutant with various constructs of viral promoter-linked myc genes induced dense foci in 3Y1 cells, an established cell line derived from rat embryo fibroblasts, whereas transfections with either species alone did not. Four cell clones were isolated from colonies grown in soft agar medium, and found to contain at least a single copy of both DNA species and to express both sequences at relatively high levels. These results indicate that the two immortalizing genes used in the present study collaborate to transform 3Y1 cells.

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Administration of 1-nitrosoindole-3-acetonitrile (NIAN) at doses of 40 to 300mg/kg body weight by gastric intubation to male F344 rats induced up to 100-fold increase in ornithine decarboxylase activity with a maximum after 24hr and up to 10-fold increase in DNA synthesis with a maximum after 16hr in the pyloric mucosa of the stomach. These results suggest that NIAN has potential tumor-promoting activity in carcinogenesis in the glandular stomach.

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Cancer incidences for major sites were compared among Koreans in Osaka, Japan, Koreans in Korea and Japanese in Osaka by calculating standardized proportional incidence ratios (SPIR's), in addition to updating the findings on cancer mortality experiences of Koreans and Japanese in Osaka reported before. Compared with Japanese, Koreans in Osaka had significantly higher mortality rates from cancers of the esophagus, liver and lung in males, and liver in females. Mortality rates among Koreans in Osaka were significantly lower for stomach cancer in both sexes and for breast cancer in females. Compared with Korean counterparts in the homeland, Koreans in Osaka had a reduced risk for cancers of the stomach in males and the uterus in females. On the other hand, an elevated risk was observed for cancers of the esophagus, colon, liver and lung among Korean males in Osaka and for cancers of the colon and liver among Korean females in Osaka. The risk for cancer of the breast in females was similar among Koreans in the host and home countries. These different cancer patterns among Koreans in the host and home countries and Japanese are discussed in relation to their life styles, such as smoking, drinking and dietary habits, which have been investigated by means of questionnaire surveys.

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The relationship between breast self-examination (BSE) and pre-treatment clinical stage of breast cancer was assessed in female breast cancer patients consisting of 30 patients practicing BSE monthly, 60 patients practicing occasionally, and 60 patients who had rarely or never practiced BSE. These patients were matched by age, residence, and hospital. More frequent practice of BSE was associated with more favorable clinical stage. The percentage of stage I patients was 33% for monthly performers, and this was significantly higher than the value of 14% for those who had rarely or never practiced. BSE practice was significantly related to tumor size measured before treatment in a dose-response manner. The average values of maximum tumor diameter were 2.5cm for monthly performers, 3.0cm for occasional performers, and 3.5cm for those who had rarely or never practiced. The results suggest that BSE practice increases the probability of detecting smaller cancers at an early stage, and this may lead to a more favorable prognosis.

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Analytical epidemiologic studies are less effective when dietary habits are homogenous within a population. For that reason, we performed chronological and spatial correlation analyses between mortality from stomach and large intestinal cancers and various foods/nutrients intakes. The age-adjusted death rates (AADRs) for male colon cancer were strongly associated with the population size and those for female colon cancer and male rectal cancer were weakly associated with the population size, but the AADRs for stomach cancer in both sexes and those for female rectal cancer were not associated. From 1969-71 to 1981-83, the AADRs for stomach and female rectal cancers decreased and the AADRs for colon and male rectal cancers increased. With an increasing population size and from the earlier period to the recent period, the intakes of western-style fat-rich foods such as butter & margarine, cheese and ham & sausage increased and those of rice, fish and some traditional Japanese foods decreased. The results of geographical correlation analyses were generally consistent with the results from the urban-rural variations. The chronological correlation analyses suggested that stomach cancer might be associated with Japanese-style dietary habits after almost no lag time and colon cancer might be associated with westernized dietary habits after a lag time of about 10 years.

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Trends in the histologic types of lung cancers during 1966-1985 by 5-year intervals were analyzed by using 4, 419 cases in the National Cancer Center Hospital, Japan. Histologically confirmed cases increased to 76.6% in the last 5-year interval. The proportions of histological types in males have remained relatively unchanged; about 40% squamous cell carcinoma, one-third adenocarcinoma, and about 10% each of large cell carcinoma, small cell carcinoma and other types. In females, an increase of adenocarcinoma from 52% to 69.1% and a decrease of squamous cell carcinoma from 21% to 11.4% were noteworthy, and the other three types amounted to less than 10% each. These trends were compared with those obtained from the Annals of Pathological Autopsy Records in Japan during 1974-1983, which contained 26, 844 lung cancer cases; the results were similar except for an increase of small cell carcinoma. The recent increase of small cell carcinoma was considered to be due to a change in diagnostic categorization, associated with a decreased rate of undifferentiated carcinoma. However, the increase of small cell carcinoma as well as adenocarcinoma in younger patients requires further study from both demographic and etiological viewpoints.

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A systematic analysis of the cellular components of intestinal metaplasia (i.e., goblet cells with or without brush border cells and Paneth cells) was performed in endoscopic gastric biopsies from 984 patients without gastric ulcer or carcinoma; 359 Swedish and 625 Japanese patients. The Japanese patients, matched for age and sex, had twice as much intestinal metaplasia as the Swedes. The frequency of goblet cells and columnar cells with brush (absorptive) border increased with increasing age in both ethnic groups. Complete intestinal metaplasia (i.e. the presence of at least two or all three cellular components) was twice as frequent in the Japanese. This may reflect differences in the environment (including food habits) of the gastric mucosa in the two populations. Structural mucosal changes (i.e. pseudo-villus formations) were found more than twice as often in the Japanese. Since pseudo-villus changes occurred as often as goblet cells and brush border cells in both groups, it is suggested that the above-mentioned mucosal structural change should be considered for inclusion among the histological criteria of intestinal metaplasia of the stomach. Incomplete intestinal metaplasia (i.e. the occurrence of goblet cells without the other cellular components) occurred in similar proportions in the two populations. The present findings support the theory that incomplete intestinal metaplasia may be a para- rather than a preneoplastic phenomenon.

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N-Butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was given to male Fischer 344 rats at a dose of 0.05% in drinking water for 2, 4, 6 and 12 weeks, and double soft agar colony formation of the uroepithelial cells was determined periodically, during and after this administration. In the group administered BBN for 2 weeks, no significant colony growth was observed until week 8. In the group given BBN for 4 weeks, colony growth was observed at week 4 and the numbers of colonies remained constant until week 8. In the group given BBN for 6 weeks, significant colony growth was observed at weeks 6 and 8. In the group on BBN for 12 weeks, colonies grew from week 4 and significant numbers of colonies were observed from week 6, increasing up to week 10. Colony formation preceded papilloma development in the rat bladder, and was dependent on the duration of BBN administration. The effect of amino acids and sodium saccharin on colony formation was also evaluated. The rats were given 0.05% BBN for 3 weeks, followed immediately by the administration for 9 weeks of 2% L-tryptophan, 1% D-tryptophan, 2% L-leucine, 2% D-leucine, 2% DL-leucine, 2% L-isoleucine, 2% DL-isoleucine or 5% sodium saccharin in the diet. At week 12, the numbers of colonies were significantly higher in the groups given sodium saccharin, L-leucine, DL-leucine, L-isoleucine, DL-isoleucine and D-tryptophan. This method provides a potentially useful approach toward analyzing the early events in bladder carcinogenesis and may be applicable to detect new bladder carcinogens and promoters.

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This investigation was designed to determine whether any lesion would be induced in germ-free animals exposed to Aspergillus versicolor alone. Twenty-one male Wistar germ-free rats bred in our Laboratory were monoassociated by A. versicolor while 20 male germ-free rats of the same strain were used as controls. All rats were maintained on our home-made pellet diet for 2 years. Animals that died or were killed were autopsied and examined pathohistologically. Eighteen of the 21 monoassociated rats (86%) had necrosis in the liver, 16 of them (76%) had foamy cell granuloma in the lung, 11 of them (52%) had fibrosis in the pancreas and 12 of them (57%) had nephron lesions in the kidney, while none of the germ-free control rats had such lesions, indicating that these lesions were induced by A. versicolor. Further, 62% of the monoassociated rats had tumors in the pleura, lung and endocrine organs, while 15% of the controls had tumors only in endocrine organs. It was noted that two of the monoassociated rats developed pleural mesotheliomas and another developed a squamous cell carcinoma in the lung, presumably due to conidiospore inhalation. This investigation showed that A. versicolor, a common mold, at a high concentration in the absence of other microbes, induced severe organ damage and some tumors in rats.

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Adult fish of an interstrain hybrid (F₁) of inbred medaka, obtained from crosses between HO4C and HB32C, were exposed for 2hr to an aqueous solution of the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine at concentrations of 15, 20, 25, 30, and 35ppm. Survival and neoplastic changes were examined over a 6-month period. A large variety of neoplasms were induced, including melanoma, papilloma, ovarian tumors, olfactory epithelioma, branchioblastoma and fibroma. More than 60% of the tumors were classified as melanoma on the basis of histological examinations. A markedly higher cumulative incidence of the melanoma with a dose-related response was demonstrated in the F₁ hybrid fish compared to the parental strains. The latent period for melanoma development, however, remained unchanged in F₁ compared to the parents. The variety of tumors induced in the F₁ fish was greater than in the parental strains. The results indicate the usefulness of F₁ hybrid fish in testing the carcinogenicity of certain water-soluble chemicals, due to their high sensitivity.

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The genetic effect of the H-2 complex on the development of chemically induced pulmonary adenomas was clearly demonstrated in H-2 congenic strains with an A/Wy background which had a high susceptibility to pulmonary adenoma. A single subcutaneous injection of either urethane or 4-nitroquinoline 1-oxide was given to nine strains of mice. Among them, the number of adenoma foci per mouse was significantly higher in A/Wy (haplotype H-2^a), A/J (H-2^a), A.TL (H-2^t1) and A.AL (H-2^a1) than in A.BY (H-2^b), A.SW (H-2^a), A.CA (H-2^t) and A.TH (H-2^t2) strains. In addition, the average number of adenoma foci in A/Wy (H-2^a) was more than 20 times that in the B10.A (H-2^a) strain. Thus, multiplicity of adenoma foci appeared to be regulated by at least two genes, one of which is located in the I or S region in the H-2 complex and the other in the non-H-2 genetic background. The genes in the H-2 complex were distinct from Pas-1 locus, which is probably identical to ptr.

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An attempt was made, by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), to induce differentiation in Ph¹-positive (PB-1049) and Ph¹-negative (LN-1049) B lymphoblastoid cell lines established from a patient with chronic myelogenous leukemia, and a Ph¹-positive line (PB-1049-T) derived from a tumor nodule formed by inoculation of PB-1049 cells into nude mice. The changes induced by TPA in all lines were consistent with differentiation towards plasma cells, and included enhanced clustering of floating cells, reduced DNA synthesis, increase of the cytoplasm/nucleus ratio and appearance of a large amount of rough endoplasmic reticulum in the cytoplasm. Immunoglobulin concentration was estimated by ELISA in the supernatant of the three cell lines. Remarkable increases were observed in PB-1049 and PB-1049-T cultures treated with TPA. These results suggest that the Ph¹-positive as well as Ph¹-negative B lymphoblastoid cell lines examined have the potential to perform the proper functions of B lymphocytes in vitro.

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The presence and location of cytochrome P-450 in Donryu rat hepatocyte culture lines, Ac2F cells and 3 other cell lines were assessed by indirect immunofluorescence examination using anti-cytochrome P-450 monoclonal antibodies. Ac2F cells and other hepatocyte cell lines were selectively stained at their nuclear envelope, but not the cytoplasm, with a monoclonal antibody selective to a high-spin form of cytochrome P-448 (P-448H), although this monoclonal antibody stained primary cultured normal rat hepatocytes at both cellular components and did not stain hepatoma cells of 2 transplantation lines. The results of unscheduled DNA synthesis assay with Ac2F cells using several carcinogenic aromatic amines (4-aminoazobenzene derivatives and amino acid pyrolysis products) suggested that this nuclear envelope-associated cytochrome P-450 activates a restricted portion of these aromatic amines, i.e., a tryptophan pyrolysis component and a glutamic acid pyrolysis component. These results indicate that rat hepatocyte culture lines lack (or contain a reduced amount of) the cytoplasmic cytochrome P-450 but maintain a characteristic type of cytochrome P-450, probably a kind of cytochrome P-448H in their nuclear envelope, and this may be involved in oxidative metabolism of a restricted portion of aromatic amines.

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The purpose of this study was to determine whether the combination of C6-1.2 monoclonal antibody (MoAb) (established in our laboratory), which is specifically reactive with Lewis lung carcinoma (3LL), and cell-wall skeleton of Propionibacterium acnes C7 (P. acnes-CWS) would significantly decrease established spontaneous metastases of 3LL. C6-1.2 MoAb combined with intratumoral (or intralesional) injection of P. acnes-CWS, when administered at an early stage of the experiment, showed a significant reduction of lung metastases in 3LL-bearing mice. The treatment modality of C6-1.2 MoAb alone did not decrease the lung tumor colonies. On the other hand, the combined treatment of intravenous injection of P. acnes-CWS and C6-1.2 MoAb did not significantly reduce the lung metastases of 3LL tumors in 3LL-bearing mice compared with the treatment of P. acnes-CWS alone. In contrast, when the primary tumor was surgically removed, the most remarkable reduction of lung metastases was observed by the combination of intravenous administration of both C6-1.2 MoAb and P. acnes-CWS. We concluded that the antimetastatic activity of C6-1.2 MoAb and P. acnes-CWS varies depending on the injection route, and also that surgical excision of the primary tumor can lead to remarkable reduction of 3LL lung metastases.

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The effects of naloxone on human ovarian cancer cell growth in vitro and in vivo were examined by using an established cell line, designated KF, derived from human ovarian carcinoma. When the KF cells were incubated in the presence of various concentrations of naloxone for 72hr, the cell proliferation was inhibited in a dose-dependent manner in the concentration range of 30 to 120μ M naloxone. The growth-inhibitory effect of naloxone could not be detected by ⁵¹Cr-release assay or colony-forming assay. The inhibition of cell proliferation by naloxone resulted from the inhibitory effect on protein synthesis in the KF cells, as confirmed by a marked decrease of incorporation of[³H]valine. In order to determine the effect of naloxone on tumor growth in vivo, ip injections 3 times a week of 2.5 or 5.0mg/kg naloxone were initiated either one week prior to tumor inoculation (pretreated groups) or one week after tumor inoculation (post-treated groups). Nude mice in the pretreated groups had significantly smaller tumor volumes than those in the untreated group as long as naloxone was administered. On the other hand, in the post-treated groups a significant growth retardation was observed 2 weeks after initiation of treatment with 2.5 mg/kg naloxone. In comparison to a median survival time of 38 days for untreated controls, the naloxone pre- and post-treated groups showed increases of 26-71% in median survival.

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