Tyramine was identified as a new mutagen precursor in Japanese soy sauce, becoming mutagenic after treatment with nitrite under acidic conditions. The mutagenic compound was identified as 4-(2-aminoethyl)-6-diazo-2, 4-cyclohexadienone, and its specific mutagenic activity was 112 revertants/μg towards Salmonella typhimurium TA100 without S9 mix.

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The effect of tumor promoters on the organization of BALB/c 3T3 fibroblasts was examined by indirect immunofluorescence microscopy using anti-actin antibodies. Potent tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate, teleocidin, dihydroteleocidin B and debromoaplysiatoxin, in spite of their different chemical structures, all induced disruption of microfilaments. This change was still observed in the presence of actinomycin D.

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The mutagenicities of twelve N-nitrosamines were tested on Salmonella typhimurium TA100 and Escherichia coli WP2 uvrA/pKM101 in the presence of rat liver S9 or hamster liver S9 by“preincubation”and“pour-plate”assays. The following eleven N-nitrosamines were mutagenic: N-nitroso-N-dimethylamine; N-nitroso-N-diethylamine; N-nitroso-N-di-n-propylamine; N-nitroso-N-di-n-butylamine; N-nitroso-N-methyl-n-amylamine; N-nitroso-piperidine; N-nitrosomorpholine; N-nitroso-N-methyl-piperazine; N-nitroso-N-methyl-benzylamine; N-nitroso-N-methyl-phenylamine and N-nitroso-N-phenyl-benzylamine. N-Nitroso-N-diphenylamine was not mutagenic. E. coli WP2 uvrA/pKM101 was more sensitive than S. typhimurium TA100 to the mutagenic actions of N-nitroso-dialkyl amines, N-nitroso-alkyl-aryl amines and N-nitroso-diaryl amines, but S. typhimurium TA100 was more sensitive to those of N-nitroso-cyclic amines. Hamster liver S9 was better than rat liver S9 for metabolic activation of N-nitrosamines, and the preincubation step enhanced the mutagenicities of N-nitrosamines.

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Incidences of duodenal tumor induced by oral administration of hydrogen peroxide (HPO) and catalase activities in the duodenal mucosa, blood and liver were correlated in C3H/HeN, C57BL/6N, (C57BL×C3H)F₁ (B6C3F₁) and hypocatalasemic C3H/C^b_smice. A solution of 0.4% HPO was given to the mice as drinking water for about 6 months. Incidences of duodenal tumor were 11.1% in C3H, 31.8% in B6C3F₁, 100% in C57BL and 91.7% in C3H/C^b_s mice. Catalase activities (10-₄k/mg protein) in the duodenal mucosa, blood and liver were 5.3, 7.8 and 75.3 in C3H, 1.7, 7.7 and 62.8 in B6C3F₁, 0.7, 5.1 and 40.7 in C57BL, and 0.4, 0.4 and 33.3 in C3H/C^b_s mice, respectively. The coefficients of correlation (r) of catalase activities with the incidences of duodenal tumor in C3H, B6C3F₁ and C57BL mice are -0.83 in duodenal mucosa, -0.98 in blood and -0.99 in liver. The results indicate that the incidence of duodenal tumor induction by HPO is dependent on the individual mouse strain and may be determined by the genes controlling the catalase activities.

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The recessive mutation lethal(2) giant larva⁴ (l(2)gl⁴) of Drosophila melanogaster causes the development of malignant tumors in the whole brain of homozygous larvae. A mutant brain fragment implanted into the abdomen of a wild-type adult female kills the host in about 10 days. Neuroblasts in situ in l(2)gl⁴ larvae showed normal karyotypes, but, when cultured in adult abdomens for one transfer generation, about 10% of the cells showed chromosome aberrations. Subculturing the neuroblasts for four transfer generations showed that malignancy (i.e., lethality to the host) as well as chromosomal abnormalities increased with time of subculture. Many virus-like particles were detected in l(2)gl⁴ neuroblasts after in vivo culture, whereas no such particles were detected in l(2)gl⁴ neuroblasts in situ in larvae. These particles contained RNAs homologous in sequence to the DNA of the movable element copia. They were indistinguishable from previously identified retrovirus-like particles in cultured Drosophila cells. It is proposed that the l(2)gl⁴ mutation reduces the genome integrity, resulting in transplantation-triggered genetic abnormalities, such as chromosomal abnormalities, increased transcription or replication of copia elements, and production of retrovirus-like particles.

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The changes of colonic epithelial ornithine decarboxylase (ODC) activity and DNA synthesis following intrarectal administration of a tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA), or various bile acids to male noninbred rats were studied. A single instillation of TPA, at a dose as low as 16nmol, led to a significant (about 10-fold) increase in colonic ODC activity. Peak ODC activity was observed at 4hr, and the enzyme activity returned to the control level about 24hr after intrarectal TPA. This pattern was almost the same as that observed after sodium deoxycholate treatment. TPA showed more potent induction of ODC activity than deoxycholate, although the maximal induction was greater in the case of deoxycholate treatment. Both TPA and deoxycholate stimulated DNA synthesis at 2 days after intrarectal instillation, after an initial depression at 4-12hr. A structure-activity study of 26 bile acids revealed that 5β-cholanoic acid with α-hydroxy groups in two of the 3α, 7α, 12α positions and 5β-cholanoic acid with a 3α-hydroxy group induced colonic ODC activity significantly, while the 3α, 6α-dihydroxy acid did not. Replacement of hydroxy groups by keto groups or a change from α to β configuration decreased the ODC-inducing activities. Tri-substituted 5β-cholanoic acid derivatives, whether hydroxy or keto, did not stimulate ODC. These data indicate that a specific bile acid structure with a definite spatial relationship of the hydroxy groups is required for induction of colonic ODC activity.

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Several clones of reconstituted cells were isolated by fusion of karyoplasts of mouse melanoma B16 cells with cytoplasts of rat myoblastic L6 cells and processed by the mica-press-adsorption method for electron microscopy. Extrachromosomal small circular DNAs of less than 1μm in contour length (smaller circular DNA) were present in both parental cells, and circles larger than 1μm (larger circular DNA) were found more frequently in reconstituted cells at an early stage after the clonal isolation. Mitochondrial DNA was not released from mitochondria by this method. The new phenotypes of reconstituted cells were stable, but larger circles were lost after prolonged cultivation of the cells. The possibility that the larger circular DNAs result from intrachromosomal DNA rearrangement induced by rat cytoplasts is discussed.

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Two partially purified fractions of the ethanol precipitate (70-95%) of the water extract of Harding-Passey mouse melanoma, which inhibit protein and DNA syntheses of B-16 melanoma cells in culture, also inhibit protein synthesis in various cell-free systems. By examining their inhibitory effects on limited reactions of protein synthesis, it was found that one of them (ME II₁) inhibits protein synthesis by blocking aminoacyl-tRNA formation, while the other (ME IV₂) does not. This inhibition of aminoacyl-tRNA formation was not limited to specific amino acids. Since the amino acid-dependent pyrophosphate (PPi)-ATP exchange reaction catalyzed by aminoacyl-tRNA synthetases was not inhibited, it was concluded that some factor(s) in ME II₁ inhibits amino acid transfer from aminoacyl-AMP to tRNA. ME II₁ contains more than 20 proteins from 10, 000 to 90, 000 daltons. EDTA treatment of this fraction caused the release of low-molecular substances with inhibitory activity from the proteins. The molecular weights of the active substances are less than 5, 000 daltons. The active low-molecular substances are apparently not peptides or nucleotides.

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Enolase isozymes (α, β and γ enolases) in the extracts of pediatric tumors (neuroblastoma, ganglioneuroblastoma, rhabdomyosarcoma and Wilms' tumor) were determined by means of enzyme immunoassay systems. All tumor tissues examined contained α enolase at high levels (2070-19100ng/mg protein). The β and γ enolases were present at high levels particularly in rhabdomyosarcoma (886±750ng/mg protein) and (ganglio)neuroblastoma (2060±890ng/mg protein), respectively. Immunohistochemical studies confirmed these results. Serum levels of these enolase isozymes were also determined in pediatric tumor patients. Before treatment, a serum sample from a patient with rhabdomyosarcoma contained a high level of β enolase and serum samples from patients with (ganglio)neuroblastoma contained high levels of γ enolase. However, the levels of serum β and γ enolases were low in patients with Wilms' tumor. The elevated level of β or γ enolase in serum from rhabdomyosarcoma or (ganglio)neuroblastoma patients was markedly decreased after adequate treatment (operation, chemotherapy or radiation). The results indicated that the enolase isozymes are useful marker antigens for differential diagnosis and therapeutic monitoring of neuroblastoma and rhabdomyosarcoma.

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Cloned cell lines with low and high metastatic potentials were established from a Lewis lung carcinoma. Those with a high metastatic potential, LM12-3-II cells, grew slower than those with a low potential, P-29 cells, both in vivo and in vitro. No significant difference between these cell lines was found in their susceptibility to natural killer cell-mediated lysis in vitro. Both LM12-3-II and P-29 cells showed poor organization of microfilament bundles containing actin. LM-12-3-II cells showed lower cloning efficiency in semisolid 0.3% and 0.6% agar medium, lower lysosomal enzyme activities and lower homotypic aggregation than P-29 cells. LM12-3-II cells adhered to monolayers of endothelial cells more slowly than P-29 cells, although they adhered to a subendothelial matrix a little more rapidly than P-29 cells. On the other hand, LM12-3-II cells adhered to the surface of plastic culture dishes more firmly than P-29 cells. The neuraminidase-accessible sialic acid of LM12-3-II cells was less than that of P-29 cells. Thus, the firmness of adhesion was positively correlated with the metastatic potential in these cells.

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Antitumor activity of Lactobacillus casei YIT 9018 (LC9018) was studied in BALB/c mice by using two syngeneic tumors; methylcholanthrene-induced tumor (Meth A fibrosarcoma) and Kirstein murine sarcoma virus-transformed BALB/3T3 (K234 tumor). Administration of an LC9018-Meth A cell mixture induced complete suppression of the tumor growth, while simultaneous injections of LC9018 and Meth A cells into different sites had no suppressive effect on the tumor growth. Administration of the mixture subsequently induced specific transplantation immunity to the challenge tumor, which started to be generated on about the 5th day after the administration and continued to at least the 30th day. Administration of an LC9018-K234 cell mixture also induced suppression of the tumor growth and generated specific antitumor immunity. Neutralization (Winn type) tests showed that T lymphocytes possessed tumor cytotoxicity but humoral immune serum did not, suggesting that the T cells with LC9018-potentiated antitumor immunity functioned in the suppression of the tumor growth.

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Studies were performed with a clonogenic cell assay system to determine the in vitro sensitivity of carcinoma of the lung to standard chemotherapeutic drugs. Formation of colonies in vitro occurred in 22 of 29 specimens (76%), including 4 of 7 squamous cell carcinomas, 11 of 16 adenocarcinomas, all of 3 large cell carcinomas, and none of 3 small cell carcinomas. Eighteen of these 22 assays (82%) showed growth which was adequate for chemosensitivity testing. Antitumor drugs tested in this study were cis-diamminedichloroplatinum, 5-fluorouracil, adriamycin, mitomycin C, vindesine, vincristine, vinblastine, bleomycin, peplomycin and l-phenylalanine mustard, which are used clinically for chemotherapy of lung cancer. Of ten drugs tested against at least two different tumors, vindesine and mitomycin C were identified as being active against more than three tumors (17%). Twelve of 18 specimens (67%) were not sensitive in vitro to any of the drugs tested. Of the remaining 6 specimens (33%) showing sensitivity to any drug, four tumors were sensitive to only one drug, one tumor was sensitive to three drugs and another was sensitive to four drugs. The in vitro chemosensitivity results seemed well correlated with clinical experience. The human tumor clonogenic cell assay system appears to be a reasonable model for the study of chemosensitivity in carcinoma of the lung.

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The antitumor effect of bestatin against syngeneic tumors such as colon adenocarcinoma 26 (colon 26) and myeloid leukemia C1498 (C1498) was investigated. Bestatin effectively inhibited the growth of both tumors in vivo. The inhibition observed for colon 26 was the largest when bestatin was administered on days 1 to 5 after transplantation of the tumor. The inhibition of C1498 was the largest when bestatin was administered on days 7 to 11. In contrast to these results, bestatin was not at all inhibitory to either tumor line in vitro. Thus, the effect of bestatin administration on the functions of the spleen cells of mice bearing tumors was examined. Spleen cells taken from mice bearing colon 26 and C1498 did not neutralize the corresponding tumor. However, when bestatin wasa dministered to these mice, spleen cells from the administered mice did neutralize the corresponding tumor. Bestatin enhanced antibody-dependent cell-mediated cytotoxicity and natural killer cell activity of the spleen cells of colon 26-bearing mice. Further, bestatin did not inhibit the growth of colon 26 in BALB/c nu/nu mice. It was concluded that bestatin exhibits its antitumor effect against colon 26 and C1498 indirectly by immunomodulation via spleen cells and possibly via T cells.

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