Male Syrian golden hamsters were given a pellet diet containing 2.0% butylated hydroxyanisole (BHA) or a powdered diet containing 1.0% BHA for 24 weeks. Papillomas of the forestomach were induced in all hamsters given either of these diets.

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The single-cell origin of bladder cancer was established in mice with cellular mosaicism for phosphoglycerate kinase (PGK). Administration of N-butyl-N-(4-hydroxybutyl)nitrosamine in the drinking water resulted in development of large, solitary, infiltrating bladder carcinomas. On electrophoresis, PGK from all the cancer tissues examined gave only a single spot.

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The effect of butylated hydroxyanisole (BHA) and ethoxyquin (EQ) on hepatocarcinogenesis initiated with N-ethyl-N-hydroxyethylnitrosamine in rats was investigated. The results clearly demonstrated that BHA and EQ inhibited the development of γ-glutamyltranspeptidase-positive foci, hyperplastic nodules and hepatocellular carcinomas in the rat system employed.

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Thiobenzamide (a thiono-sulfur-containing xenobiotic), when administered to male Sprague-Dawley rats primed with a single low dose of diethylnitrosamine, enhances the number and size of both γ-glutamyltranspeptidase-positive hepatocellular foci and cholangiofibrotic areas. Its effect seems to be greater than that of the known promoter phenobarbital.

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Mutagenic components in commercial beef extract and in cooked ground beef were adsorbed from their aqueous solutions on cotton bearing covalently linked trisulfo-copper-phthalocyanine residues (blue-cotton). By repeating the adsorption and elution, efficient concentration of the mutagenic components with a satisfactory overall recovery was achieved. Carboxymethyl cellulose column chromatography was found to be an excellent means to separate 2-amino-3, 8-dimethylimidazo-[4, 5-f]quinoxaline (MeIQx) from 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ), two strong mutagens that have previously been found in heated beef samples. Chromatography of the mutagenic components of beef extract on this column gave two mutagenic fractions which corresponded to MeIQx and IQ in elution profile. In reversed-phase high performance liquid chromatography, the major active component of the MeIQx fraction and that of the IQ fraction behaved identically with standard samples of MeIQx and IQ, respectively. The contents of these mutagens in a sample of Difco beef extract were estimated at 200-300ng of MeIQx and 20-40ng of IQ per gram. By the same fractionation procedures, mutagenic substances in the cooked beef were fractionated into MeIQx-type and IQ-type components. The activity distribution among these two fractions was similar to that found for beef extract.

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Mutagenicity of 3'-methyl-N, N-dimethyl-4-aminoazobenzene (3'-Me-DAB) and its N-demethylated products was examined using rat liver 9, 000g supernatant fraction (S-9) together with Salmonella typhimurium TA98 or TA100 as a tester strain. The expression of mutagenicity of 3'-Me-DAB required the presence of both microsomes and cytosol as sources of enzymes as well as NADPH as a cofactor. 3'-Me-DAB and its N-demethylated products showed no mutagenicity towards either strain when preincubated with S-9 from untreated rat livers. The involvement of a cytochrome P-450 species in the mutagenic activation was demonstrated with hepatic S-9 by using specific enzyme inducers and inhibitors. The treatment of rats with polychlorinated biphenyls or 3-methylcholanthrene resulted in a marked increase in the ability of S-9 to activate these compounds, whereas phenobarbital induction was not effective. All the mutagenic activities were considerably decreased by adding cytochrome c to the S-9 mixture, but the activation was insensitive to 1-(1-naphthyl)-2-thiourea and methimazole, high-affinity flavin-containing monooxygenase substrates. Carbon monoxide, metyrapone, and 2-diethylaminoethyl-2, 2-diphenylvalerate hydrochloride, potent cytochrome P-450 inhibitors, had no inhibitory effect on the mutagenic activation. In contrast, 7, 8-benzoflavone, a specific inhibitor of cytochrome P-448, considerably inhibited the reaction. These results suggest that cytochrome P-448 and a cytosol component are involved in the mutagenic activation of 3'-Me-DAB and its N-demethylated products.

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Four groups were used, each consisting of 24 male ACI rats. Goup I received a single subcutaneous injection of 20mg 1, 2-dimethyihydrazine (DMH)/kg body weight and was fed on basal diet until the termination of the experiment. Group II received the same single injection as in group I and then, starting one week later, was fed the diet containing 1% dextran sulfate sodium (DS-M-1). Group III received an injection of 0.9% sodium chloride and then DS-M-1 diet. The control group was given neither DMH nor DS-M-1 diet. The experiment was terminated after 40 weeks. A significantly higher incidence of colorectal carcinoma was observed in group II, as compared with group I (P<0.02). These data demonstrate the enhancing effect of DS-M-1 on colorectal carcinogenesis.

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Rat acinar cells in the azaserine-induced hyperplastic nodules or adenomas in the siderotic pancreas produced by repeated iron injections were found to be resistant to iron accumulation. These iron-resistant acinar cell lesions coincided rather well with the lesions having markedly decreased activity of γ-glutamyl transpeptidase. These properties of the acinar cells could be useful for the identification of early lesions in pancreatic carcinogenesis.

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From MDCC-MSB1, the original Marek's disease virus-associated lymphoblastoid cell line, two sublines named MDCC-MSB1-33C and MDCC-MSB1-41C were differentiated. The MDCC-MSB1-33C cell line grew well at 33°, but grew poorly and finally stopped growing at 41°. On the other hand, the MDCC-MSB1-41C cell line grew actively at 41°, but at 33°, continuous cultivation was difficult to achieve. MDCC-MSB1-33C grew suspended in the liquid medium, as did the original producer MDCC-MSB1 cell line, while MDCC-MSB1-41C grew attached to the substrate. Moreover, in soft agar, MDCC-MSB1-33C formed loose colonies, but MDCC-MSB1-41C formed packed colonies. Neither Marek's disease viral antigens nor herpes-type particles could be detected in either subline, although a Marek's disease tumor-associated surface antigen was found on the surface of both.

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All Friend leukemia cell lines induced by polycythemic strains of Friend leukemia virus complex express an appreciable amount of spleen focus-forming virus (SFFV)-coding envelope gene (env)-related glycoprotein with a molecular weight of 55 kilodaltons (gp55). A clonal, highly differentiation-inducible Friend leukemia cell line, T3-C1-2-O(2-O), possesses not only gp55 but also gp51 as SFFV-specific gene products. The peptide map of gp51 is quite similar to that of gp55 and the difference in their molecular weights is primarily dependent on their oligosaccharide content. In the differentiation-induced state, gp51 becomes far less detectable than gp55 in 2-O cells. The biological significance of this novel SFFV-coding glycoprotein is discussed.

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Neutral glycolipids and gangliosides of four clonal variants of rat fibrosarcoma AS-653 cells with different transplantability werea nalyzed. A highly malignant clone A had a much lower quantity of GM3 ganglioside and a much higher quantity of lactosylceramide as compared to clones Z and G, which showed low transplantability and less malignancy, and a variant clone P, which showed tumorigenicity only in ascites form. A similar correlation was found with the quantity of an unidentified slow-migrating neutral glycolipid in various clones. This glycolipid was present in trace amount in the original highly malignant clone A, increased moderately in clones G and Z, and increased greatly in clone P, which showed no subcutaneous transplantability. The results of these studies suggestthat a blockage of synthesis of GM3 ganglioside and a long chain neutral glycolipid occurred with enhanced malignancy, and an enhanced synthesis of GM3 ganglioside and the long chain neutral glycolipid is associated with a decrease in transplantability.

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Rat prostatic tumor (Dunning R 3327 tumor) is a well-differentiated adenocarcinoma of Copenhagen strain rat; its growth is extremely slow and androgen-dependent. Two new sublines (Chiba University A and B; CUA, CUB) were obtained during passages; CUA, squamous cell carcinoma, grew at moderate speed, and CUB grew rapidly, being composed of spindle-shaped cells and large bizarre polygonal cells. Growth of CUA was slightly androgen-dependent, while that of CUB was independent of sex hormones. Activities of acid phosphatase were increased in both CUA and CUB as compared with the original R 3327, so the less differentiated sublines of CUA and CUB showed unusual progression. In contrast, activities of alkaline phosphatase in CUA and CUB were diminished as compared with that of the original tumor. Both androgen and estrogen receptors were detected in the cytosol from R 3327, but there was no detectable amount of androgen and estrogen receptors in CUA. CUB did not show any androgen binding but estrogen binding was observed in the cytosol, though estrogen did not have any detectable effect on the growth of this tumor.

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Renal cell carcinoma tissues from two patients, one with and one without erythrocytosis, were successfully transplanted into athymic nude mice. Transplantations of the erythrocytic tumor through six successive generations of nude mice produced a significant (P<0.001) elevation in mean hematocrit from 36.5±2.1% (range 32-42%) to 53.7±5.1% (range 40-63%), in comparison with a non-erythrocytic tumor which showed a progressive fall in hematocrit from 46.5±2.0% (range 41-50%) to 36.8±1.6% (range 33-40%). Non-grafted control nude mice maintained stable hematocrit levels from an initial level of 45±0.5% to 46.5±1.2% when studied over the same time interval. Similarly red cell mass values in the mice transplanted with the erythrocytic tumor (5.04±1.85ml/100g) were considerably higher than in both the non-grafted nude mice (3.39±0.81ml/100g) and the non-erythrocytic tumor-grafted mice (3.8±0.3ml/100g) after 6 generations of transplants. Plasma erythropoietin levels in the erythrocytic tumor-grafted mice (169.4±83.1mU/ml) were significantly (^P<0.02) higher than in the non-grafted controls (22.2±9.5mU/ml), and furthermore the erythropoietin levels in the tumor extracts were significantly (^P<0.02) higher in the tumors from erythrocytic mice (range 54.7 to 234.6mU/g tumor) than in the tumors from non-erythrocytic mice (range 0.3 to 1.9mU/g tumor). In vitro monolayer cultures of these tumors confirmed the higher erythropoietin levels in the erythrocytic renal carcinoma (138mU/ml) as compared with culture media of non-erythrocytic tumors (15-91mU/ml) using the fetal mouse liver assay ⁵⁹Fe incorp. into heme). The present studies indicate autonomous erythropoietin production by human renal cell carcinomas both in vivo in nude mice and in vitro in tissue cultures.

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The objective of this investigation was to characterize non-cloned neuroblastoma cells immunohistochemically. In this study, the cholinergic cells in three mouse tumors were identified by using a choline acetyltransferase (CAT) antibody. Different cholinergic cell distribution patterns were found in the three tumors by using a fluorescence-activated cell sorter (FACS). An antibody to CAT was prepared by immunization of guinea pigs with CAT-antigen from bovine brain. The specificity of the antibody obtained was examined with mouse cervical spinal cord. Identification of the cholinergic cells was performed in three types of non-cloned tumor culture cells, neuroblastoma C-1300 in A/J mice, glioblastoma 203GL in C57BL6 mice and lymphosarcoma 6C3HED in CBA mice. The CAT content and distribution were determined by radiochemical assay, immunohistochemical staining, and individual cell counting with a FACS. The results of radiochemical assay and immunohistochemical staining were in agreement with the CAT distribution pattern determined by cell counting with FACS IV.

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The roles of macrophages in preventing metastasis of a homotransplantable lymphoma were studied in Syrian golden hamsters. Removal of the primary tumor led to enhanced development of metastatic foci. Macrophage activities as demonstrated by macrophage spreading and cytostatic activity were augmented in tumor-bearing hosts, but depressed in tumor-resected hosts. Metastases were facilitated by treatment with silica, an agent that blocks macrophage functions. These results may suggest that immune-activated macrophages contribute to the inhibition of metastases. Metastases were completely inhibited by local irradiation of the primary tumors but such an effect was abolished by the resection of irradiated tumors. Irradiated tumor cells may work as potent immunogens to augment the generation of immune-activated macrophages.

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Cytotoxic T lymphocytes (CTL) were raised against syngeneic plasmacytoma MOPC-315 cells by culturing spleen cells immunized with MOPC-315 cells 7 to 14 days previously, either in the presence or absence of MOPC-315 cells. The characteristics of these CTL were investigated in relation to those of natural killer (NK) cells. A portion of the CTL population cross-reacted with NK-sensitive YAC-1 cells. The cytotoxic activity of these CTL was blocked strongly by D-mannose and weakly by α-methyl-D-mannoside and glucose, but was not affected by galactose or N-acetyl-D-glucosamine. All of the above compounds blocked the activity of NK, but not that of CTL against allogeneic cells. Thus, these CTL have some similar properties to NK cells, but are clearly distinct from NK cells in the surface phenotype: asialo GM₁ is positive in the membrane of NK, but negative in that of CTL. The mannose-sensitivity of the CTL activity was not observed when CTL to MOPC-315 cells were generated during the culture of twice-immunized spleen cells plus MOPC-315 cells. Furthermore, the activity of CTL obtained during the reculture of mannose-sensitive CTL with MOPC-315 cells was not blocked by mannose. These results suggest the existence of a maturation process of the CTL population to syngeneic MOPC-315 cells in terms of the sensitivity to D-mannose.

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Cytotoxic T lymphocytes (CTL) were raised against syngeneic plasmacytoma MOPC-315 cells by culturing spleen cells immunized with MOPC-315 cells 7 to 14 days previously, in the presence of MOPC-315 cells for 5 days. The cytotoxic activity of these CTL was blocked by D-mannose, indicating that mannose-containing carbohydrate moieties are involved in the expression of cytotoxic activity. Investigations were performed to determine which cells, effector or target, possess carbohydrate moieties, by employing procedures by which a part or most of the carbohydrate on cells can be removed. When target cells were treated with 2-deoxy-D-glucose, tunicamycin or trypsin, the levels of cytotoxic activity of the CTL were reduced. However, the reduced activities were still blocked by adding D-mannose at the effector phase. Treatment with α-mannosidase did not affect the level of cytotoxicity or mannosesensitivity. In contrast, when effector cells were treated with tunicamycin by adding it to the culture one day before harvest, the level of cytotoxic activity was reduced, but the cytotoxic activity was no longer blocked by D-mannose. A similar result was obtained when effector cells were treated with periodate after 5 days of culture. However, the treatment of effector cells with α-mannosidase did not affect the cytotoxicity of the CTL. Thus, it was shown that effector cells possess mannose-containing carbohydrate moieties which are involved in the full expression of CTL to syngeneic MOPC-315 cells.

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Nine animal lectins, i.e., Sarcophaga peregrina agglutinin, Balanus roseus agglutinin, Aplysia kurodai agglutinin, Balanus balanoides agglutinin, Tetraclita aquamosa japonica agglutinin, Misgurnus anguillicaudatus lectin, Asterina pectinifera agglutinin, Helix aspersa agglutinin and Helix pomatia agglutinin, were tested for induction of cytolysis mediated by polymorphonuclear leukocytes. Among them, S. peregrina agglutinin and B. roseus agglutinin lysed murine target cells in co-operation with polymorphonuclear leukocytes (PMNs) from the peritoneal cavity of mice. PMNs can lyse various tumor cells in the presence of S. peregrina agglutinin, although normal spleen cells were also lysed. This lectin-dependent cytolysis by PMNs was inhibited by galactose, a sugar which is specifically recognized by S. peregrina agglutinin. S. peregrina and B. roseus agglutinins were inhibitory to in vivo development of MM46 tumor cells. These results suggest that PMNs can lyse various target cells in the presence of appropriate animal lectins and that some animal lectins participate in tumor rejection.

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Cytotoxic T lymphocytes (CTL) against autologous fresh osteosarcoma cells were generated in mixed lymphocyte tumor culture (MLTC), and then were propagated and maintained for up to 19 days with culture medium containing T cell growth factor (TCGF). CTL generated with MLTC and propagated with TCGF (TCGF-CL) lysed fresh autologous osteosarcoma cells, but not autologous phytohemagglutinin-blastoid lymphocytes. More than 90% of the propagated lymphoblastoid cells were E-rosette forming cells. A subpopulation of TCGF-CL was Leu-2a positive and Leu-3a negative CTL. The cytotoxic activity to the autologous tumor detected by ⁵¹Cr release assay was not blocked by cold allogeneic tumor cells from 4 out of 5 patients tested. Human leukocyte antigen and chromosomes of TCGF-CL showed no abnormality.

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Two hundred and seventy-one sera from patients with lung cancer were checked to determine whether they exerted a blocking or potentiating effect on normal lymphocyte-cytotoxicity against lung cancer targets using a microcytotoxicity assay. A blocking effect was observed in 15 of 49 untreated patients (30.6%), but a potentiating effect was detected in 10 of 49 (20.4%). Such effects were not correlated with stages of the disease or the clinical status of patients. Such a blocking effect was also detected in patients with pneumonia, pulmonary tuberculsois, benign mediastinal tumor and malignancies other than lung cancer, but the potentiating effect could not detected in such patients. Using an indirect membrane immunofluorescence test, IgG antibodies against QG-56 of a lung cancer cell line were detected in 54.3% of potentiating sera and in 20.5% of blocking sera. Such antibodies were cross-reactive with 2 other lung cancer cell lines and one of 4 uterine cancer lines, however, no cross-reaction was observed against 3 breast cancer lines, 3 of 4 uterine cancer lines, one malignant melanoma line and one pancreas cancer line. The effector cells exerting the potentiating effect were identified as non-adherent, Fc receptor-bearing lymphocytes. Therefore, the potentiating effect might be typical antibody-dependent cell-mediated cytotoxicity.

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Antitumor activity of bleomycin against Ehrlich ascites carcinoma in mice was examined in the presence and absence of a polyene antibiotic, pimaricin. A single intraperitoneal (ip) injection of bleomycin and pimaricin in combination into tumor-bearing mice on day 1 produced a synergistic life-prolongation effect as compared with that of each agent alone. Accumulation of abdominal ascites was almost completely blocked by bleomycin in combination with the tetraene antibiotic. Frequent ip injection of small doses of bleomycin and pimaricin on day 1 produced a much greater increase in life span than the single ip injection. To obtain effective synergism under these treatment schedules, it appeared that the dose of bleomycin should be reciprocally proportional to the dose of pimaricin.

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