Recent studies of gastric cancer among Hawaii Japanese yield clues which might explain the disappearance of this tumor from the American scene. The frequency of stomach cancer is decreasing among second generation Hawaii Japanese, although high rates persist in the first generation. This decrease is probably not the result of improved diagnostic or treatment methods, but can be explained on the basis of a decreased consumption of traditional Japanese food, which is rich in salt and nitrate, accompanied by increased use of fresh fruit and vegetables. This supports the hypothesis that invasive cancer is the last of a sequence of steps which begins as a mutation of gastric mucosa to an intestinal form. This is caused by the presence in the stomach of mutagenic nitroso compounds derived from the nitrosation of dietary amines, a chemical event which may be blocked by ascorbic acid. The intestinalized gastric mucosa is at increased risk of both ulcer and cancer, which explains the similarity of the epidemiologic background of the two conditions.
Differences in the male: female ratio of gastric cancer between Japanese and Caucasians, as well as the greater frequency of the diffuse type of gastric cancer among Japanese patients at all age levels, suggest that there is a strong host influence upon the morphology and behavior of this cancer once induction has occurred. Hawaii Japanese demonstrate better survival with both the diffuse and intestinal types of gastric carcinoma than do Hawaiians or Caucasians in Hawaii, probably because they have fewer tumors at stage IV at the time of diagnosis. The fact that all three races have a similar proportion of stage I tumors indicates that the excess of disseminated cancers among the non-Japanese probably cannot be explained on the basis of differences in the diagnostic methods.

View full abstract

Effect of methylnitrosocyanamide (MNC) on a cultured mammalian cell line was examined in comparison with that of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). It was observed that MNC was about 3 times more potent than MNNG in the experiments on toxicity and inducibility of chromosome aberration and mutation. An increase in toxicity of MNNG was observed by minimizing the elevation of pH, while toxicity of MNC was more potent in the serum-free condition than in the presence of serum. Further, MNC was shown to be extremely rapid-acting, that is, 10min was enough to exert its toxic effect.

View full abstract

Antitumor activity of 6-thioguanine against L-1210 leukemia was studied in combination with various antitumor agents in clinical stages, such as mitomycin-C, carbazilquinone, cyclophosphamide, 3, 3'-dimesyloxydipropylamine tosylate (864T), 4-hydroperoxyisophosphamide, and 3-[(4-amino-2-methyl-5-pyrimi-dinyl) methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU). The combination treatments with 6-thioguanine and each of six agents, especially with ACNU, showed a distinct therapeutic effect against the early L-1210 leukemia at dosage levels not producing any significant antitumor activity with each agent alone (ip-ip). The excellent antitumor activity of the combination of 6-thioguanine with ACNU was also proved in various sites of tumor inoculation and routes of drug administration systems, i. e., ip-iv, iv-ip, and iv-iv systems. Moreover, the effectiveness of the combination of these two drugs was clearly shown against advanced L-1210 leukemia which was refractory to each agent alone.

View full abstract

5-Fluorouracil inhibited DNA synthesis markedly using various DNA precursors such as deoxyuridine, orotic acid, uracil, and uridine except for thymidine. 2-(Tetrahydrofuryl)-5-fluorouracil (FT-207) did not inhibit DNA synthesis with any of the precursors tested. The metabolisms of 5-fluorouracil and FT-207 in mice and rats were studied. When administered intravenously 5-fluorouracil was rapidly degraded to fluoro-β-alanine in both mice and rats, while at most 70% of FT-207 was slowly degraded after a prolonged period. The metabolites of FT-207 were found to be 5-fluorouracil and fluoro-β-alanine in both species of animals. In vitro degradation of FT-207 into 5-fluorouracil was observed mainly in the microsomal fraction in the presence of NADPH. This result suggested that microsomal electron-transport system was concerned with the degradation of FT-207.

View full abstract

The possibility of passive immunotherapy was examined using syngeneic antitumor serum, which could lyse tumor cells in co-operation with immune or activated macrophages. Intraperitoneal injection of syngeneic antiserum into mice soon after inoculation of MM46 tumor cells clearly suppressed peritoneal or subcutaneous tumor growth. However, the antiserum did not suppress an established tumor. The tumor-specific synantibody responsible for the protection was shown by gel filtration to be in the IgG fraction, not the IgM fraction.

View full abstract

The possibility of passive therapy of 6∼8-day established tumors with syngeneic antitumor antiserum was tested using (1) combination of the antiserum and immunopotentiators, (2) combination of the antiserum and inflammatory agents, and (3) repeated injections of antiserum. Therapeutic effect on an ascites tumor was seen using antiserum in combination with BCG or lipopolysaccharide, or less clearly with carrageenan. Combinations of antiserum and PS-K, histamine, or croton oil did not have a synergistic therapeutic effect, but repeated injection of a small amount of antiserum did have a therapeutic effect on established peritoneal and subcutaneous tumors.

View full abstract

Polymethylene-bis (1-nitrosourea), polymethylene-bis (1-nitroso-3-nitroguani-dine), and polymethylene-bis (1-nitroso-p-toluenesulfonamide) derivatives were tested for antitumor effect against rat ascites hepatoma AH-13 and mouse leukemia L-1210. Bisnitrosoureas were effective against AH-13 and L-1210, bisnitrosoguanidines were effective against AH-13 alone, and bisnitrosotoluene-sulfonamides were ineffective against both tumor lines. Of all these compounds, 1, 1'-ethylene-bis (1-nitrosourea) (EBNU) was the most effective.
The antitumor effect of EBNU was compared with that of 1, 3-bis (2-chloroethyl)-1-nitrosourea (BCNU). Intraperitoneal administration of EBNU according to the schedule, day 1, days 1 and 5, and days 1, 5, and 9 after intraperitoneal inoculation of L-1210 showed marked prolongation of host survival, although the effective doses used were a few times higher than those used in BCNU to obtain a similar effect. The minimum effective dose (MED) of EBNU on AH-13 cells was estimated as 1mg/kg, which was 10 times less than that of BCNU, suggesting that EBNU was more effective than BCNU against AH-13.

View full abstract

Human gastric cancer-associated antigens were solubilized from fresh ascites and surgical specimens by 3M KCl extraction. The antigenicity of these extracts was measured by leucocyte migration inhibition assay with an improved modification devised in this laboratory. Antigens extracted mutually reacted with autologous and allogeneic leucocytes taken from cancer patients.
Three out of 11 (27%) patients and 7 out of 28 (25%) patients with various cancers showed positive migration index values against 13 ascites tumor cell antigens in the autologous and allogeneic reaction systems, respectively. Reactions were positive in 4 out of 10 patients with gastric cancer against autologous antigens, and in 10 out of 26 (38%) of the patients against allogeneic tumor antigens.
On the other hand, one patient with gastric cancer reacted well with various sources of antigens extracted from allogeneic cells of gastric cancer, while the other patient with gastric cancer did not react with any of five gastric cancer antigens tested so far. These findings suggested that the cell-mediated immune response of patients with gastric cancer against autologous and allogeneic tumor antigens extracted was detectable in the test system. It is considered at present that the 3M KCl antigen may not be relate to human leucocyte antigen, and some common antigenic reactants capable of reacting with several gastric cancer may be involved in this reaction.

View full abstract

Tumor-specific immunity mediated by thymus-derived lymphocytes (T cells) was established in C3H/He mice against syngeneic X5563 plasmacytoma. Splenic T cells from mice, which exhibited resistance to tumor challenge, revealed a potent in vivo tumor-neutralizing activity as well as in vitro cytotoxicity. In contrast, spleen cells from mice 7 days after tumor cell inoculation (7-day tumorbearing mice) exhibited no protective activity when assayed by in vivo tumorneutralization test, whereas these cells exhibited almost comparable in vitro cytotoxic activity to that from the tumor-resistant mice. Any suppressor cell activity was not detected in 7-day tumor-bearing animals. While a slight in vivo protective activity was observed in the spleen cells from 14-day tumor-bearing mice, this activity was still significantly weaker than that of spleen cells from mice similarly inoculated with tumor 14 days before but whose tumor had been removed 7 days before the assay. The development of in vivo protective immunity in tumor-resected mice was suppressed by intravenous inoculation with 7000R X-irradiated tumor cells.
These results indicate that in vitro reactivity of immune T lymphocyte population is not always correlated with in vivo protective immunity, but there is a substantial decrease in in vivo immune capability of T cells from tumor-bearing animals, and that this suppression may be ascribed to the presence of a large amount of tumor-associated transplantation antigens rather than to suppressor cell activity.

View full abstract

The mode of generation of free radicals of daunomycin, adriamycin, and carboquone in the NADPH-rat liver microsome system was studied at room temperature by electron spin resonance (ESR) spectroscopy. ESR signals of all these quinoid, anticancer chemicals were detected when dissolved oxygen in the reaction mixture was consumed since the radicals are easilyaut oxidizable. All the radicals had an appreciable lifetime under anaerobic conditions. However, there were differences in the mode of their generation between daunomycin and adriamycin, on the one hand, and carboquone, on the other, with respect to the lag time and the effect of the amount of chemicals, pH of the medium, kind of electron donors, NADPH and NADH, and the presence of excess of DNA. Especially, ESR signal reappeared after the first signal had decreased considerably, in the case of daunomycin and adriamycin but not in carboquone.
Intact Ehrlich ascites tumor cells also gave rise to an ESR signal of adriamycin and carboquone, but the former signal was prevented from appearing in the presence of glucose.

View full abstract

Induction of 8-azaguanine-resistant mutation, DNA single-strand breaks, and chromosome aberrations by treatment with ethyl methanesulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), nitrogen mustard hydrochloride (HN₂), or 4-nitroquinoline 1-oxide (4-NQO) was compared under similar experimental conditions using cultured FM3A cells from a C3H mouse mammary carcinoma.
All the chemicals induced 8-azaguanine-resistant mutations and chromosome aberrations in a dose-dependent manner; a good correlation between the two activities was demonstrated. An alkaline sucrose gradient method demonstrated that DNA single-strand breaks were induced only in a high dose range by 1- and 24-hr treatment with the chemicals. One exception was that a 1-hr treatment with HN₂ produced an anomalous sedimentation pattern. These data suggest that caution is necessary for the interpretation of results obtained by the alkaline sucrose gradient analysis, and that examination of mutagenicity and chromosome aberrations is more feasible as screening procedures for potential mutagens than analysis of DNA single-strand breaks.

View full abstract

Mutagenicity of various mycotoxins and the efficiency of mutagenic mycotoxins in producing DNA single-strand breaks and chromosome aberrations were examined using a mammalian cell line. It was found that aflatoxin-B₁, mycophenolic acid, patulin, penicillic acid, and sterigmatocystin induced 8-azaguanine-resistant mutations. Aflatoxin-B₁, mycophenolic acid, and sterigmatocystin had little effect on DNA single-strand at high concentrations. In the treatment with patulin and penicillic acid, severe breaks were found at higher concentration than at the concentration where the mutation was induced. Incidence of chromosome aberrations by the treatment with these mycotoxins correlated fairly well with their mutagenic activity.
Chaetoglobosin-B, fusarenon-X, (-) luteoskyrin, and ochratoxin-A did not induce 8-azaguanine-resistant mutation.

View full abstract

Examinations were made on the carcinogenic effects of a chemical compound, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and a physical carcinogen, whole-body irradiation with fission neutrons, on the gastrointestinal tract of male albino Sprague-Dawley rats. The carcinogens were used singly and together in order to investigate their possible synergistic effects on the induction of adenocarcinomas of the stomach and small intestine. Of the 13 animals treated with the chemical, MNNG, and living more than 9 months, 9 showed gross tumors (5 gastric and 4 duodenal), confirming the high incidence of gastrointestinal carcinomas induced by MNNG in the rat. There were no gastrointestinal tumors found after neutron exposure. When the 2 carcinogens were combined, no additivity or synergism occurred. After neutron irradiation, a dental syndrome with loss of incisor teeth was observed. The effect of neutron irradiation on subgingival pathology of the teeth is being investigated.

View full abstract

Activities and isozyme patterns of acid phosphatase and esterase were studied in rat liver at different intervals after partial hepatectomy to clarify the grade of immaturity of normal regenerating liver cells as a control for the unlimited proliferation of hepatoma cells.
Acid phosphatase and esterase activities in the liver were elevated during a 12-hr period after hepatectomy, while their isozyme patterns did not change from those of immature liver. Similar findings were also observed in the liver of sham-operated rats.
Eighteen hours after the operation, at the S phase before cell division, the isozyme pattern of these enzymes began to shift from an adult liver-type to an immature one resembling those of the infant liver 3 weeks after birth rather than those of newborn or fetal liver. Two or three days after partial hepatectomy, the isozymes characteristic of an immature liver type were more apparent. Although enzyme activities mostly returned to the normal adult level one week after the operation, the isozyme patterns did not completely return to those of an adult liver.
These results indicate that despite the rapid proliferation of liver cells, the grade of cell differentiation of the regenerating liver after partial hepatectomy is much nearer to that of the normal adult liver rather than that of the fetal liver.

View full abstract

Although the ascitic fluid of animals bearing various tumors and that of mice induced by complete Freund's adjuvant had high activity for inducing differentiation of myeloid leukemic cell line (Ml) from an SL mouse to macrophages and granulocyte-like cells, the activity in the ascitic fluid of syngeneic mice bearing the Ml cells was markedly reduced. Macrophages and granulocytes were abundant in the active ascites of animals bearing tumors (8 to 12% of the total ascites cells) while in the ascites of syngeneic mice bearing the Ml cells they were not (0.1 to 0.7% of the total ascites cells). Appearance of lymphocytes in the ascites of both types was not significantly different. Although the conditioned media of the Ehrlich tumor cells, Ml cells, and whole ascites cells with the Ml cells were not active in inducing differentiation of the Ml cells, the conditioned media of all the ascites cells with Ehrlich tumor cells and those of peritoneal macrophages and granulocytes in mice did show a high activity. These results indicate that the peritoneal macrophages and granulocytes in the ascites are responsible for the production of factors stimulating differentiation of the Ml cells.

View full abstract

Biological, histological, and ultrastructural studies were made on tumors produced in weanling hamsters inoculated with hamster embryo fibroblasts (HEF) transformed by herpes simplex virus type 2 or with cultured cells (tumor cell lines T₁ and T₂) derived from the tumor tissues. The malignancy of transformants increased through passages in vitro and in vivo. Histologically, two tumor cell lines (155-4T₁ and U-15T₁) produced fibrosarcomas and one (U-26T₁) produced lesions resembling "malignant fibrous histiocytomas." Ultrastructurally, fibrosarcomas produced by 155-4T₁ and U-15T₁ consisted mainly of fibroblast-like cells with extracellular collagen fibers, whereas malignant fibrous histiocytoma-like lesions produced by U-26T₁ consisted of undifferentiated cells, multinuclear giant cells, histiocyte-like cells, and fibroblast-like cells. Frequently, various kinds of "nuclear bodies" were found in the nuclei of tumor cells. Several herpes virus-like particles (120∼140nm in diameter) were detected in some nuclei of undifferentiated tumor cells produced by U-26T₁.

View full abstract

The isozymes (enzymes I and III) of branched-chain amino acid transaminase (EC 2.6.1.42) from various human tissues were separated by DEAE-cellulose column chromatography. Their distributions were found to be considerably different from those in rat tissues reported before. In rats, all tissues examined contained enzyme I and in addition enzyme II was found in the liver and enzyme III in the brain, ovary, and placenta. In humans, however, enzyme II was not found in any tissues examined including the liver, and enzyme III was found in many other tissues besides brain, ovary, and placenta. All normal human tissues except the lung, ovary, and brain contain less enzyme III than enzyme I.
Various human cancers of the liver, kidney, stomach, pancreas, and uterus showed significantly higher ratios of enzyme III to enzyme I than those of the corresponding normal tissues. Fetal liver and kidney also contained much higher concentrations of enzyme III than adult liver and kidney. These findings suggest that change of the isozyme pattern of this enzyme in cancer is similar in humans and rats, and that cancer tissues tend to express more immature phenotypes than normal tissues.
Enzymes I and III of human tissues showed the same substrate specificities for valine, leucine, and isoleucine, and these amino acids competed for the active site of the enzyme. Thus the hereditary diseases, hypervalinemia and hyperisoleucine-leucinemia, may be due to genetic alteration of the enzyme protein, resulting in change of its substrate specificity.

View full abstract

The pelvic region of random bred female ICR-JCL mice and CF₁ mice was irradiated with various doses of X-rays at 1-week intervals to determine the relationship between the X-ray dose and induction of rectal carcinoma. The incidence of rectal carcinoma in ICR mice was zero after a single dose of 2, 000 rad of X-rays, but 31% after a single dose of 3, 000 rad, 6% after 2 doses of 1, 500 rad, 25% after 3 doses of 1, 500 rad, 42% after 2 doses of 2, 000 rad, and 95% after 3 doses of 2, 000 rad. This cancer developed in 70% of CF₁ mice exposed to 2 doses of 2, 000 rad. No case of this tumor was observed in the control animals not exposed to X-rays. The development of this tumor was found to depend on the X-ray exposure dose. Local X-irradiation of the pelvic region is one of the effective methods for inducing rectal carcinoma in mice.
Rectal cancers induced by X-irradiation were adenocarcinoma of the tubular, papillary, and mucinous type, and frequently showed invasive growth into the deep layers of the rectal wall.

View full abstract

Colchicine and vincristine sulphate differentiate sharply between hepatocyte mitosis associated with the Ehrlich ascites tumour, and regenerative (post-CCl₄ necrosis) hepatocyte mitosis. In the former case there is a gross depression of prophase index and a marked lowering of the overall mitotic index. In the latter, although a degree of prophase depression is evident, this is insufficient to prevent an overall substantial elevation of mitotic index due to accumulation at metaphase. Very low doses of colchicine claimed to be effective in stathmokinesis of hepatocytes under influence of hepatomitogenic tumour brei and tumour extracts, produced no discernible effect on the mitotic pattern of hepatocytes in Ehrlich ascites tumour-bearing mice nor in post-CCl₄ regenerating livers.
The results are discussed in the context of known features of tumour-associated hepatocellular mitosis.

View full abstract

New anthracycline antibiotics have been isolated from the culture of Streptomyces galilaeus MA144-Ml. Among 14 anthracycline compounds, aclacinomycin-A showed the strongest activity in inhibiting leukemia L-1210 and had lower toxicity than others. Antitumor activity of aclacinomycin-A against leukemia L-1210 and P-388, solid sarcoma-180, and lymphosarcoma 6C3HED was examined in comparison with adriamycin and daunomycin. Aclacinomycin-A showed the same degree of activity against leukemia L-1210 and P-388, when administered intraperitoneally, as daunomycin and somewhat less than adriamycin. In oral administration, aclacinomycin-A also exhibited a significant activity on leukemia L-1210. The degree of inhibition of the growth of sarcoma-180 and 6C3HED lymphosarcoma transplanted subcutaneously by aclacinomycin-A was almost the same as that of adriamycin and daunomycin, although the optimal dose was about twice more than adriamycin.
Acute cardiotoxicity of aclacinomycin-A by a test using hamsters was more than 10 times lower than that of adriamycin.

View full abstract

A new line of tissue culture cells derived from a slow-growing hepatoma 8999 was established and named 8999C. The isolation method, growth pattern, and morphology of the 8999C cells are described. Several hepatic enzyme activities in 8999C cells were compared to those in the original hepatoma 8999. The ornithine aminotransferase (EC 2.6.1.13), tyrosine aminotransferase (EC 2.6.1.5), and arginase (EC 3.5.3.1) activities in the 8999C cells were one-third, one-tenth, and one-hundredth of those of the respective activities in the original hepatoma 8999, and mitochondrial serine protease, which has much higher activity in hepatoma 8999 than in normal liver cells, was not detected in 8999C cells. Tyrosine aminotransferase in 8999C cells was induced by dexamethasone but not by N⁶, O²'-dibutyryladenosine 3', 5'-cyclic monophosphate or insulin. Unlike in hepatoma 8999, glucocorticoid did not induce arginase in 8999C cells.

View full abstract

The acid-soluble components of mouse-liver cytosol, prepared at timed intervals after intragastric administration of a single carcinogenic dose of ¹⁴C-dimethylnitrosamine, were separated by column chromatography. The columns were calibrated with known in vitro metabolites of the nitrosamine, and the elution profiles were compared with those of the mouse-liver system. The results suggest that the ¹⁴C-methyl label is transferred to many of the same compounds as identified in vitro, but that numerous other labeled compounds are also present. The possible significance of these metabolites to the pathogenic processes induced by dimethylnitrosamine has yet to be determined.

View full abstract

Tumor-inhibitory effect of combined use of mitomycin-C and streptococcal preparation (OK-432) or yeast cell wall (YCW) was examined. Twenty-four hours after intraperitoneal inoculation of Ehrlich carcinoma cells, combination therapy was carried out and tumor growth was observed for 40 days. About one-half of mice treated with yeast cell wall at a single dose of 1mg survived free of tumor. Mitomycin-C at a single dose of 2μg was not effective. However, in combination with yeast cell wall, tumor suppression was observed in 70% of the mice. This tumor-inhibitory effect was enhanced by subsequent treatment with OK-432 or yeast cell wall. When these materials were injected separately or in combination with mitomycin-C, the number of peritoneal exudate cells increased about 3 to 6 times after 3 days and these cells exhibited cytotoxic effect on tumor cells. Hemagglutinating antibody to sheep erythrocytes was hardly affected by the combination therapy.

View full abstract