Smoked dry bonito (katsuobushi), an everyday food item for most Japanese people, was found to contain 2-amino-3, 8-dimethylimidazo [4, 5-f]quinoxaline (MeIQx), the content of which was estimated at about 2ng/g. This content is similar to the known MeIQx content of cooked beef. The katsuobushi also contained another mutagenic component, the total activity of which was 1/6-1/3 that of the MeIQx. This component was similar to 2-amino-3, 4, 8-trimethylimidazo[4, 5-f]quinoxaline (4, 8-DiMeIQx) with respect to its behavior in high-pressure liquid chromatography and its ultraviolet absorption spectrum.

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The effects of glycerol administration on pulmonary tumorigenesis induced by 4-nitroquinoline 1-oxide (4NQO) were examined in male ddY mice, that were given 5% glycerol solution instead of drinking water after a subcutaneous injection of 4NQO. The incidence of pulmonary tumor-bearing mice and the mean number of induced tumors per mouse were significantly enhanced in mice given glycerol after 4NQO treatment, compared with mice given 4NQO alone. The results demonstrate effects including that of promotion by glycerol in 4NQO-induced pulmonary tumorigenesis.

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The mutagenic potential of seven carcinogenic N-nitrosopropylamines was examined by means of Ames' preincubation assay using liver 9000g superanatant (S9) fractions from rats, hamsters, mice, rabbits, monkeys and humans for metabolic activation. N-Nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP), N-nitrosobis(2-oxopropyl)amine (BOP), N-nitrosobis(2-acetoxypropyl)amine (BAP), N-nitroso-2, 6-dimethylmorpholine, N-nitrosomethyl(2-hydroxypropyl)amine (MHP) and N-nitro-somethyl(2-oxopropyl)amine all showed positive mutagenicity in strain TA100 in the presence of liver S9 from each of the uninduced animals, but N-nitrosobis(2-hydroxypropyl) amine was negative. The mutagenic activity of MHP was highest with liver S9 from the hamster, but that of BAP was lowest with hamster liver S9. With regard to the activities of the other N-nitrosopropylamines, there were no significant differences among five animal species. In the presence of liver S9 from humans, HPOP, BOP and MHP showed positive mutagenicity. With the exception of HPOP and BOP, the animal or human liver S9-mediated mutagenicity of these N-nitrosopropylamines was almost completely lost upon removal of NADP⁺ from the assay system, preincubation in an atmosphere of carbon monoxide, or addition of cytochrome cto the S9 mixture. Metyrapone decreased the activities of five compounds (except for BOP) by between 29 and 71%, whereas 7, 8-benzoflavone was totally lacking in this effect. These results demonstrated that the phenobarbital-inducible major cytochrome P-450 in animal and human livers is involved in the mutagenic activation of the N-nitrosopropylamines.

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Ethanol, potassium metabisulfite, formaldehyde and hydrogen peroxide were tested for tumor-promoting activity in a two-stage stomach carcinogenesis experiment. Male outbred Wistar rats were given N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the drinking water (100mg/liter) and a diet supplemented with 10% sodium chloride for 8 weeks. Thereafter, they were maintained on drinking water containing either 10% ethanol, 1% potassium metabisulfite, 0.5% formalin (formaldehyde) or 1% hydrogen peroxide for 32 weeks and then sacrificed for necropsy and histological examination. In the pylorus of the glandular stomach, potassium metabisulfite and formaldehyde significantly increased the incidence of adenocarcinoma after initiation with MNNG and sodium chloride. Hydrogen peroxide did not enhance the tumor yield, and ethanol showed a tendency to decrease neoplastic development. In the forestomach the incidence of squamous cell papilloma was significantly increased in the groups given hydrogen peroxide or formaldehyde, irrespective of prior initiation. Duodenal adenocarcinoma was induced by the initiation alone (10%) and the incidence was not affected by the subsequent treatments. The results indicate that potassium metabisulfite and formaldehyde both exert tumor-promoting activity in the rat glandular stomach.

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Hepatic metabolism in vitro of dimethylnitrosamine (DMN), diethylnitrosamine (DEN), 3, 4-benzopyrene (BP) and 7, 12-dimethylbenz[a]anthracene (DMBA) was assayed using the liver of rats maintained for 3 weeks on liquid diets containing three levels of carbohydrate (CHO) with or without supplementation of ethanol (5g/100ml). Ethanol consumption increased the metabolism of DMN and DEN to different degrees depending on the diet consumed simultaneously. Lowered CHO intake, which by itself increased the metabolism, dose-dependently augmented the increase due to ethanol: the lower the CHO intake, the more remarkable was the enhancement caused by ethanol. Ethanol increased microsomal cytochrome P-450 content only when combined with low-CHO diets. It was confirmed by a Salmonella mutagenicity test that the enhancement of DMN and DEN metabolism was accompanied by increased capacity of the liver to activate these nitrosamines to mutagens. On the other hand, neither lowered CHO intake, nor ethanol consumption, nor their combination affected the metabolism of BP and DMBA. Ethanol combined with a diet deficient in CHO even suppressed the metabolism of BP.

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Changes in the level of expression of glucose-6-phosphate dehydrogenase (G6PD) within propyl nitrosamine-induced preneoplastic and neoplastic lesions in F344 rats and Syrian golden hamsters were investigated using an immunohistochemical approach. Previously demonstrated increases in G6PD activity in rat liver and hamster pancreatic foci of altered cells were revealed as being due to elevation in the quantity of enzyme protein, suggesting an underlying change in gene expression. Furthermore, strong positive binding of G6PD antibody in thyroid, lung, urinary bladder and kidney lesions indicated that increase in this enzyme protein might be a common marker for neoplastic alteration, regardless of organ. While the function of elevated G6PD may be related to growth requirements, the finding that preneoplastic lesions in some cases bind more strongly than more malignant populations suggests additional involvement of the enzyme in other biochemical pathway(s) relevant to tumorigenesis.

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γ-Glutamyltransferase (G-GT) has been widely used as a marker of the preneoplastic stage of chemical carcinogenesis. We obtained male rats of Donryu strain that showed a reduced response to 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) in terms of induction of G-GT activity in the liver, by giving rats of this strain diet containing the carcinogen for several generations. In these animals, hepatic G-GT activity was only slightly higher than normal even after more than 20 weeks of continuous post-weaning exposure to the carcinogen, and it decreased to below the normal level if administration of the carcinogen was discontinued. In normal male rats, administration of diet containing 3'-MeDAB for more than 2 weeks resulted in an increase in hepatic G-GT activity, and the activity was increased further by a single injection of hydrocortisone. This response to glucocorticoid was also lost in “3'-MeDAB-resistant rats.” Transient elevation of fetal hepatic G-GT activity occurred at the end of gestation of “resistant rats” as well as normal rats, but the highest activity at birth of the “resistant rats” was significantly lower than that of normal rats. We observed retardation of tumor development in the liver of fourth and fifth generation rats maintained continuously on diet containing 3'-MeDAB.

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The presence of human epidermal growth factor (hEGF) was studied in a total of 210 gastric carcinomas comprising 52 early carcinomas, 113 advanced carcinomas and 45 scirrhous carcinomas. An immunohistochemical study revealed no hEGF-immunoreactivity in early gastric carcinomas, while hEGF-positive tumor cells were detected in 24 (21.2%) of the 113 advanced carcinomas and in 15 (33.3%) of the 45 scirrhous carcinomas. The incidence of hEGF-immunoreactivity in well-differentiated adenocarcinomas was significantly higher than that in poorly differentiated adenocarcinomas (P<0.05). Moreover, hEGF-immunoreactive tumor cells were observed in 13 (30.4%) of the 42 scirrhous poorly differentiated adenocarcinomas, the incidence being significantly higher than that in non-scirrhous poorly differentiated adenocarcinomas (P<0.05). The average hEGF content in the tumor tissue estimated by radioimmunoassay was 3.77±0.61 (mean±SE) ng/g wet weight in immunohistochemical hEGF-positive tumors and 2.19±0.18ng/g wet weight in hEGF-negative tumors, the difference being significant (P<0.05). Patients with hEGF-positive carcinomas (excluding scirrhous carcinomas) had much worse prognosis than those with hEGF-negative carcinomas. These results suggest that EGF produced by tumor cells plays an important role in the invasive growth and productive fibrosis of gastric carcinoma and also serves as a biologic marker of high malignancy in patients with gastric cancers.

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The analbuminemic rat is a mutant in which albumin mRNA processing is blocked owing to a seven-base-pair deletion in the 9th intron of the albumin gene. A small amount of albumin is detected in the serum of this rat. Production of serum albumin in the liver of this mutant rat was studied by an immunohistochemical method. The results showed that a few albumin-positive cells were present in the liver of the mutant rat, whereas all the hepatocytes in normal rat liver were albumin-positive. In the mutant the number of albumin-positive cells increased with age: their frequency was 10^-5 at birth and 6×10^-3 at 45 weeks of age. On administration of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) and 2-acetylaminofluorene, albumin-positive cells increased dramatically. Their numbers were 7 to 10 times those of untreated rats after 10 to 16 weeks of carcinogen treatment. The number of albumin-positive cells did not decrease after 3'-Me-DAB feeding was discontinued. In regenerating liver after partial hepatectomy, clusters of albumin-positive cells increased while the number of singlet albumin-positive cells decreased. These observations suggest that the appearance of albumin-positive cells in analbuminemic rat liver may be due to a somatic mutation of the hepatocytes.

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Placental alkaline phosphatase (PLAP)-specific monoclonal antibody (MAb) 11-D-10, which did not react with other isoenzymes of alkaline phosphatase (AP), was raised by a hybridoma technique. MAb 11-D-10 was radiolabeled and administered to athymic mice bearing human choriocarcinoma containing PLAP. This antibody was found to be more specifically localized in tumor tissue as compared to normal tissues. The tissue-to-blood ratio (T/B ratio) of MAb 11-D-10 in tumor tissue increased from 1.38 at 2 days to 2.51 at 5 days after administration. On the other hand, the T/B ratios of isotype control non-immunized IgM in tumor tissue were 0.72 and 0.87 at 2 days and 5 days after administration, respectively. ¹³¹I-labeled MAb 11-D-10 was administered to athymic mice bearing choriocarcinomas of various sizes and various PLAP contents to examine the effect on the radioimage of the differences in tumor size and PLAP content. Tumors less than 0.3cm in diameter could be imaged clearly by gamma-scintigraphy without blood pool image subtraction. The strength of the radioimage correlated fairly well with PLAP content.

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A cell line designated GBK-1 was established from a patient with anaplastic carcinoma of the gallbladder, marked neutrophilia and fever, and has been propagated for the past 18 months. The cells grew as a monolayer sheet with a doubling time of 43hr. The GBK-1 cells were of a pleomorphic polygonal epitheloid shape and they were transplantable into nude mice. Mice bearing the tumor developed marked granulocytosis. In the culture supernatant of GBK-1 cells, high colony stimulating factor (CSF) activity was evident with both human bone marrow cells and C57BL mouse bone marrow cells, and granulocytic colonies, macrophage colonies or granulocytemacrophage mixed colonies were produced. The CSF activity was distributed in the molecular weight range of 25, 000 to 60, 000 with two distinct peaks at molecular weights of approximately 50, 000 and 30, 000. CSF activity was inactivated by heat treatment at 70° for 30min. GBK-1 is a new human cell line that produces heat-labile human GM-CSF.

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To examine whether plasminogen activator reflects the functional state of estrogen receptors in human breast cancer, the enzyme activities were determined in extracts prepared from 160 breast cancer specimens and compared on qualitative and quantitative bases with the levels of steroid receptors, such as cytoplasmic estrogen receptor (ERC), progesterone receptor (PgR) and nuclear estrogen receptor (ERN). With any receptor, plasminogen activator activity was significantly higher in receptor-positive tumors than in receptor-negative tumors. When these breast tumors were categorized into 8 groups in terms of combinations of receptor status, breast cancers which were positive for all these receptors were found to contain the highest plasminogen activator activity. Furthermore, quantitative analyses demonstrated positive correlations of the enzyme activity with either ERC content (correlation coefficient +0.37, P<0.001) or PgR content (correlation coefficient +0.45, P<0.001). These results strongly suggest that plasminogen activator can be used as an effective functional marker for hormone dependence in human breast cancer.

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The present study demonstrates the intratumoral infiltration of lymphocytes mediating anti-tumor delayed-type-hypersensitivity (DTH) responses as well as in vivo protective immunity. The surface phenotype and tumor specificity of these effector lymphocytes were determined. X5563 tumor-infiltrating lymphoid cells were obtained from the tumor mass of syngeneic C3H/HeN mice 2 weeks after the intradermal inoculation of 10⁶ viable X5563 tumor cells. These lymphoid cells consisted of Thy-1-positive (29-35%), surface immunoglobulin-positive (16-29%), large granule-positive (15-25%) and esterase-staining-positive (10-20%) cells. They were tested for anti-X5563 DTH responses by utilizing a local adoptive transfer system and for tumorneutralizing activity in a Winn assay. The results indicate that X5563 tumor-infiltrating lymphoid cells exhibited appreciable anti-X5563 DTH responses and conveyed complete protection against the tumor. Treatment of these lymphoid cells with anti-Thy-1.2 or anti-Lyt antibodies plus complement revealed that the in vivo anti-tumor immune responses were mediated predominantly by a Lyt-1⁺2^- T cell subset. Such Lyt-1⁺2^- T cell-mediated immunity was tumor-specific, since X5563-infiltrating and syngeneic MH134 hepatoma-infiltrating cells exhibited DTH response and tumorneutralizing activity selectively against the respective tumor cell types. Thus, these results indicate that tumor-specific in vivo-protective Lyt-1⁺2^- T cells infiltrate into the tumor mass. The results are discussed in the context of 1) the interrelation of DTH responses and the tumor protection mediated by the Lyt-1⁺2^- T cell subset and 2) possible cellular interactions between Lyt-1⁺2^- T cells and co-existing nonspecific tumoricidal effector cells such as macrophages.

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To ascertain the clinical predictability and, in the long run, the usefulness of the human tumor/nude mouse model as a screening tool for antitumor agents, it seems particularly important to use as many tumor lines as possible and to evaluate the therapeutic effectiveness of antitumor agents in terms of the overall response rate of a range of tumors. In this study, using 11 strains of established human gastric tumor xenografts with various histological characteristics and proliferation behavior, the experimental response rates to 8 typical antitumor drugs (mitomycin C, cyclophosphamide, ACNU, cisplatin, adriamycin, vincristine, vinblastine and 5-fluorouracil) were determined and compared with the clinical values. The experimental response rates to adriamycin, vincristine and 5-fluorouracil were in good accordance with the clinical results. However, with the other drugs, significantly higher response rates were observed with the nude mouse model as compared to clinical therapy, indicating that this model tends to overestimate the responsiveness of human tumor to a number of antitumor agents. These results strongly suggest the importance of using appropriate dose levels in the nude mouse to reproduce clinically equivalent effects in this model.

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Twelve monomeric or dimeric alkaloids from Vinca rosea Linn., which had been reported to have little or no antitumor activity, were investigated to determine their combined effects with either vincristine or daunorubicin on in vitro cell growth of a P388 subline resistant to vincristine and cross-resistant to anthracyclines. We found that the combinations at subcytotoxic concentrations induced significant growth inhibition of the resistant cells, but not of the sensitive cells. Of the alkaloids examined, catharine, vindoline, catharanthine, vincarodine, and lochnerine were able to bring about complete inhibition of cell growth. Further in vitro study using vindoline revealed that at 10μg/ml it was able to completely reverse not only resistance to vincristine but also cross-resistance to vinblastine, daunorubicin, and adriamycin. In addition, we found that vinca alkaloids active in reversing resistance possess potent activities to enhance the net uptake of not only vincristine but also daunorubicin by the resistant cells, and this effect was proved to result from their inhibitory action on the active efflux process. These results provide further support for our hypothesis that both anthracyclines and vinca alkaloids can inhibit their own efflux process by interacting with the cell membrane, and this similarity provides a basis for their reciprocal cross-resistance, irrespective of their different chemical structures.

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The intracellular uptake, retention and cytotoxic effect of adriamycin (ADR) were investigated by a flow cytometry technique with NIH 3T3 cells. The intracellular uptake and cytotoxic effect of ADR increased as a function of exposure time and external drug concentration at 37°. A good correlation was found between eventual survival and intracellular ADR uptake. The intracellular uptake and cytotoxic effect of ADR increased with increasing temperature and were shown to be functions of both incubation time and temperature. The intracellular uptake and cytotoxic effect of ADR were increased even at 39° and 41°, temperatures which had no effect on the viability of cells. The combination of hyperthermia (43°) and ADR showed a synergistic effect. It was found that the cytotoxic effects of ADR in combination with various levels of hyperthermia were stronger than those which would be predicted from the intracellular ADR uptake at 37°. The degree of enhancement was temperature-dependent. Since the efflux of intracellular ADR was the same with or without hyperthermia, the increased intracellular ADR uptake caused by elevated temperature was considered to result from an increase in influx.

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Stimulation by recombinant murine interferon-gamma (rMuIFN-γ) of host defenses against the growth of Lewis lung adenocarcinoma, cell line 3LL, in the lung was examined. 3LL cells were resistant to in vitro antiproliferative activities of rMuIFN-γ. On the other hand, rMuIFN-γ augmented the killer activities of the non-adherent population of spleen cells and peripheral blood mononuclear cells (PBMC) in vivo, and the IFN also stimulated the cytostatic activities of peritoneal and splenic macrophages, but not alveolar macrophages. The combination of rMuIFN-γ with LPS synergistically activated macrophages from the lung, as well as the peritoneal cavity and the spleen, to become both cytostatic and cytolytic against 3LL cells. The macrophages activated in vitro by simultaneous incubation with these agents markedly suppressed the growth of 3LL cells in vivo. The growth in the lung of 3LL cells implanted iv was significantly (P<0.05) suppressed by combination therapy with rMuIFN-γ and LPS, but not by either agent alone. These results indicate that the potency of rMuIFN-γ action against 3LL cells can be augmented by combination with LPS, mainly through synergistic macrophage activation.

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