Fourteen human primary stomach cancer tissues were screened by Southern blot hybridization using six oncogene probes (myc, myb, H-ras, K-ras, abl, mos), and an amplification of c-myc oncogene was found in one tissue. This is the first report of c-onc amplification in primary stomach cancer tissue.

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Two alleles of c-Ha-ras-1 in a human melanoma could be distinguished by restriction fragment length polymorphism, and one of them was demonstrated to be amplified 10-20 times by hybridization to a specific probe. Molecular cloning and nucleotide sequence analysis revealed that the amplified allele contained the activated c-Ha-ras-1. In the ras family, amplification of c-Ki- ras-2 and c-Ha-ras-1 has been demonstrated in several human tumors. However, the present result provides the first direct evidence that gene amplification occurs in the activated allele.

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Human proto-oncogene c-yes-1 homologous to the viral oncogene (v-yes) of an avian Y73 sarcoma virus was mapped to region q21.3 of chromosome 18 by in situ hybridization. This finding confirmed and more rigidly substantiated the previous assignment which was based on analyses of human×mouse somatic cell hybrids and Southern blots.

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A new human cell line named FL-18, carrying a 14;18 translocation [t(14;18)(q32;q21)], was established from a Japanese patient with follicular small cleaved cell lymphoma. The established FL-18 cells had monoclonal surface immunoglobulins (IgG-κ) and were negative for Epstein-Barr virus nuclear antigens. The high-resolution banding technique indicated the breakpoints of chromosomes 14 and 18 involved in the characteristic 14;18 translocation to be at sub-bands 14q32.3 and 18q21.3, respectively. The FL-18 cell line should be useful for studying oncogenic events associated with 14;18 translocation and karyotype evolution.

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Peripheral blood cells and bone marrow cells from patients with various types of leukemia, but not those from healthy persons, were brightly immunofluorescent (IF) after staining with monoclonal antibody reactive to O-phosphotyrosine. All the IF-positive cells observed in peripheral blood were judged to be leukemic in morphology, and the IF-positive cells comprised 40-90% of total leukemic cells present in blood. Similar bright fluorescence was observed in the K562 human leukemic cell line. The results raisethe possibility that leukemic cells can be distinguished from normal hematologic cells by their increased contents of phosphorylated tyrosine residues.

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The carcinogenicity of the mutagenic compound 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ), identified in fried and broiled foods, was investigated in female Sprague-Dawley rats. Administration by gavage of IQ (0.4mmol/kg body weight) on a weekly regimen for 31 weeks starting with 6-week-old animals induced a high yield of multiple mammary gland carcinomas in a limited bioassay lasting a total of 52 weeks. In a positive control group receiving 4-aminobiphenyl (4-AB), the expected carcinomas in the mammary gland developed. IQ, but not 4-AB, also yielded neoplasms in the ear duct and to a lesser degree, in the liver, pancreas, and urinary bladder. In addition, with IQ, a high incidence of preneoplastic lesions in the pancreas was observed. Vehicle and untreated controls did not have any lesions. These findings demonstrate that IQ is a powerful multipotent carcinogen in the rat.

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The susceptibility of analbununemic rats (Nagase analbuminemic rats; NAR) to induction of bladder cancer by oral N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) was studied. Male NAR and Sprague-Dawley rats (SD) were fed normal diet or normal diet supplemented with 0.2% FANFT for 10 weeks and killed in week 52. Bladder cancer was found in 5 of 24 SD (21%) but not in 18 NAR (0%). Proliferative lesions of the bladder epithelium, which are considered to be precancerous or more advanced than precancerous changes, were found in 16 of 24 SD (67%), but in only 4 of 18 NAR (22%). NAR showed lower susceptibility than control SD to induction of bladder cancer by FANFT.

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Dose-response studies were undertaken to investigate the enhancing activity of potassium bromate (KBrO₃), a food additive, on renal tumorigenesis initiated by N-ethyl-N-hydroxyethylnitrosamine (EHEN). A total of 180 male 6-week-old F344 rats were divided into 12 groups. EHEN was given in the drinking water for the first 2 weeks at a concentration of 500ppm for initiation of carcinogenesis. Thereafter, the rats were treated orally either with KBrO₃ at a concentration of 500, 250, 125, 60, 30 or 15ppm, or with potassium bromide (KBr) at a concentration of 1750 or 350ppm for 24 weeks. The mean numbers of kidney dysplastic foci were significantly increased in a doserelated manner in rats treated with more than 30ppm KBrO₃. The mean number of renal cell tumors was significantly higher after treatment with KBrO₃ at the highest concentration of 500ppm. On the other hand, KBr had no effect. It was concluded that KBrO₃ at doses higher than 30ppm in the drinking water has an enhancing effect on renal tumorigenesis.

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Tubular adenomas developed spontaneously in ovaries of (WB×C57BL/6)F₁-Sl/Sl^t and-W/W^v mice after 150 days of age. The uteri of the W/W^vmice were hypoplastic before development of tubular adenomas, but the uteri of the Sl/Sl^t mice were not. Since oophorectomy significantly reduced the uterus weight in the Sl/Sl^t mice, we investigated the androgen-and estrogen-producing activity of Sl/Sl^t and W/W^v mice before development of tubular adenomas. Fragments of ovaries were cultured for 48 hr in serum-free medium containing either progesterone or 4-androstene-3, 17-dione (androstenedione). The amount of androgens produced from progesterone by ovaries of Sl/Sl^t mice in the presence of human chorionic gonadotropin was comparable to that of W/W^v mice. In contrast, the amount of estrogens produced from androstenedione by ovaries of Sl/Sl^t mice in the presence of follicle stimulating hormone was much greater than that of W/W^v mice. Moreover, the amount of estrogens produced was much greater than the amount of androgens in the ovaries of Sl/Sl^t mice. Therefore, before development of tubular adenomas, estrogens rather than androgens seem to be a major uterotropic hormone produced by ovaries of Sl/Sl^t mice.

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The induction of cell differentiation by a combination of 1, 25-dihydroxyvitamin D₃ [1, 25-(OH)₂D₃], recombinant gamma-interferon (rec γ-IFN), and a lipopolysaccharide from E. coli (LPS) was studied in a clonal population (clone-9) of human promyelocytic HL-60 leukemia cells in vitro. Treatment of clone-9 cells with 10^-9 to 10^-7M 1, 25-(OH)₂D₃ yielded a macrophage cell differentiation. The addition of 10 or 100U/ml of γ-IFN and 2 or 10μg/ml LPS caused a further increase in expression of the different differentiation markers. The most pronounced effects involved increases in cell attachment to the surface of tissue-culture Petri dishes and in lysozyme, nonspecific esterase, and cytolytic activities. The combined treatment with 1, 25-(OH)₂D₃ and rec γ-IFN and LPS also caused an increase in the percent of multinucleated giant cells. These results indicate the effectiveness of combining different agents in inducing cell differentiation in HL-60 cells. A similar approach may be useful in controlling myeloid leukemias in vivo.

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Hepatitis B surface antigen (HBs-Ag) was detected histologically (by victoria blue-nuclear fast red staining) in the liver in 20-40% of liver cirrhosis and/or hepatocellular carcinoma (hepatoma) cases. In hepatoma cases, HBs-Ag was usually found in non-cancerous areas of the liver. However, some HBs-Ag positive cells were also found dispersed in cancerous areas; these were regarded as HBs-Ag-infected noncancerous hepatic cells remaining undestroyed. There were a few cases where inconsistency was found between the results of the immunofluorescence technique using anti HBs-Ag serum and victoria blue staining in detecting HBs-Ag. This phenomenon was rare in non-cancerous areas, but was relatively frequent in cancerous areas.

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Highly purified recombinant human interferon-γ produced by Escherichia coli (ReIFN-γ) was examined for ability to stimulate natural killer (NK) cell cytotoxic activity and antibody-dependent cell-mediated cytotoxicity in human peripheral blood lymphocytes (PBLs) in comparison with natural human interferon-γ (IFN-γ) and with recombinant human interferon-β produced by E. coli (ReIFN-β). The activity of ReIFN-γ to stimulate NK cell cytotoxicity was about 10 times greater than that of ReIFN-β and similar to that of IFN-γ when PBLs were pretreated with each interferon for 16hr. The effects of ReIFN-γ were completely neutralized by a rabbit anti-ReIFN-γ antibody, but not by a rabbit anti-ReIFN-β. ReIFN-γ did not show any significant NK-enhancing effect when it was added directly to the killing phase of standard ⁵¹Cr-release assay for 4hr, while ReIFN-β showed a marked effect. The time course study showed that the NK-activating effect of ReIFN-γ was not expressed until 2hr after the start of incubation, while that of ReIFN-β was expressed rapidly and sufficiently within 15min. This kinetic difference between ReIFN-γ and ReIFN-β was confirmed by an experiment using the Ca²⁺-pulse method, in which the killing rate in the Ca²⁺-dependent stage of NK cytolysis was increased by ReIFN-γ, but only after 6hr of preincubation, while the effect by ReIFN-β was expressed rapidly at 30min. These findings suggest that ReIFN-γ and ReIFN-β may stimulate NK cell cytotoxicity through different intracellular events after they bind to their receptors on the surface of NK cells.

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The effect of phorbol esters on the induction of cytotoxic T cells was studied in vitro. 12-O-Tetradecanoyl phorbol-13-acetate (TPA) suppressed the induction of hapten-reactive cytotoxic T cells when added to the in vitro culture. Other phorbol derivatives such as phorbol, phorbol-l3-monoacetate and phorbol-l2-monomyristate had no suppressive activity. The suppressive activity of TPA was not due to either a cytotoxic effect or a change of the kinetics of the cytotoxic T cell development. The suppressive activity was evident when TPA was added at the early stage of the induction culture of cytotoxic T cells, but not at the later stage. Furthermore, TPA did not suppress the effector phase of cytotoxic T cell action on the target cells. A brief treatment of spleen cells with TPA induced suppressor T cell activity. This suppressive activity of TPA was correlated with the stimulation activity of lymphocytes. Thus, TPA stimulates the proliferative T cell response on the one hand and suppresses the induction of cytotoxic T cells on the other. The mechanism of the suppression of induction of cytotoxic T cells by TPA and the role of tumor promoters in carcinogenesis are discussed.

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Several types of combination therapy, including OK432, cyclophosphamide (CY), and/or lentinan plus bacterial lipopolysaccharide (LPS), reported to have strong antitumor activity against some tumors, were only slightly effective on Lewis lung carcinoma (LC) in C57BL/6 mice. Combination therapy by intralesional injection of OK432 followed by intraperitoneal administration of CY, lentinan and LPS caused almost complete regression of intradermal solid-type LC. Maximal antitumor activity was observed when lentinan and LPS were administered later than CY. Mice in which LC had regressed due to this combination therapy showed an antitumor delayed hypersensitivity reaction (measured by the footpad test), but they were not resistant to rechallenge with LC. These results provide a new model for combination therapy against weakly immunogenic tumors.

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The antitumor activity of partially purified rabbit tumor necrosis factor (TNF) in combination with lentinan or chemotherapeutic agents was tested. Partially purified TNF was obtained from TNF-containing rabbit sera by salt precipitation and ion-exchange chromatography. Its specific activity, determined in vitro as its cytotoxic activity against L929 cells in the presence of actinomycin D, was 10⁵U/mg protein or about 30-fold that of the crude rabbit sera. The growths of MH134 hepatoma in C3H/He mice and Lewis lung carcinoma in C57BL/6 mice were partially inhibited by intratumoral administration of a suboptimal dose of the TNF preparation. Additional treatment with lentinan, actinomycin D, mitomycin C or adriamycin significantly increased the antitumor activity. In particular, combination therapy with TNF and lentinan was very effective against MH134 hepatoma without having detectable side-effects, showing that lentinan expanded the therapeutical potential of TNF. These results are discussed in relation to our previous results on combination antitumor therapy.

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Several anticancer drugs were tested for induction of cytolysis mediated by poly-morphonuclear leukocytes (PMNs). Among them, actinomycin D and vincristine sulfate induced tumor lysis mediated by PMNs (drug-dependent cellular cytotoxicity). The cell-free supernatant of PMNs incubated with actinomycin D in vitro could lyse various murine tumor cells, although normal spleen cells and sheep red blood cells were not lysed. The cytolytic activity of the supernatant was resistant to treatment with superoxide dismutase, catalase, trypsin inhibitor or arginine. However, the cytolytic activity was labile to heat treatment at 56°(30min) or trypsin treatment, suggesting a protein nature. These results suggest that PMNs can lyse tumor cells in the presence of certain anticancer drugs and that a factor(s) from PMNs may participate in the killing of tumor cells.

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5'-Deoxy-5-fluorouridine (5'-DFUR) was evaluated for antitumor activity against four murine tumors (L1210 leukemia, P388 leukemia, Lewis lung carcinoma, and B16 melanoma) and a human mammary carcinoma (MX-1) xenografted in athymic mice. Intraperitoneal administration of 5'-DFUR was ineffective against B16 melanoma implanted intraperitoneally and showed less marked antitumor activity against P388 and L1210 leukemias implanted intraperitoneally or intravenously as compared with that of 5-fluorouracil (5-FU) or 1-(2-tetrahydrofuryl)-5-fluorouracil (FT-207), while oral administration of 5'-DFUR showed a similar or superior antitumor activity to that of 5-FU or FT-207 against L1210 leukemia implanted subcutaneously. 5'-DFUR showed a marked antitumor activity against MX-1 implanted subcutaneously and also showed slight antitumor activity against Lewis lung carcinoma implanted subcutaneously, while 5-FU and FT-207 did not show any significant antitumor activity against these tumors. These results suggest that 5'-DFUR may be worthy of clinical trial against solid tumors, especially cancers of the breast.

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TA-077, 1-(2-chloroethyl)-3-isobutyl-3-(β-maltosyl)-1-nitrosourea, is a new water-soluble nitrosourea derivative which is disubstituted at the N-3 position of the structure, and is activated by a unique mechanism whereby organic isocyanates can never be produced. In order to clarify the biological functions of the organic isocyanates which common nitrosourea derivatives generate, TA-077 was tested in BDF₁, mice for lethality, weight loss and hematological toxicity. TA-077 showed qualitatively and quantitatively similar toxicity to other nitrosourea derivatives which generate organic isocyanates, such as 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloro-ethyl)-1-nitrosourea (ACNU), methyl 6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-α-D-glucopyranoside (MCNU), and 1-(2-chloroethyl)-3-(β-D-glucopyranosyl)-1-nitrosourea (GANU), indicating that the organic isocyanates do not play an important role in the biological activity of the nitrosourea derivatives. Furthermore, the toxicity of TA-077 did not increase significantly (except for weight loss) when the drug was administered daily for five days, and TA-077 given according to this schedule showed far more effective antitumor activity than when given as a single treatment. These results suggested that TA-077 is an analog suitable for consecutive administration in cancer treatment.

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Antitumor activity of the whole cell and slime of an aquatic sheathed bacterium, Sphaerotilus natans IAM 12068, against ascites form of Ehrlich carcinoma in ddY mice was investigated. Intraperitoneal injection of whole cells and the slime fraction showed remarkable antitumor activity against mice inoculated with 10⁴ to 10⁵ tumor cells, the slime fraction being more effective. To examine the chemical nature of the active principle in the slime fraction, separation by Sepharose 4B gel filtration was carried out and two fractions designated as GF-P-1 and GF-P-2, which are mainly composed of protein, carbohydrate, and lipid, were obtained. GF-P-1 fraction, which contains large amounts of fucose and unidentified sugar as neutral sugar, showed marked antitumor activity at half the dose of the slime fraction, whereas the antitumor activity of GF-P-2, which is composed mainly of protein, was weak. This finding indicates that GF-P-1 fraction of S. natans slime may be a main active principle. The consistently demonstrable antitumor activity of GF-P-1 was abrogated by treatment of mice with silica, an anti-macrophage agent, suggesting that the antitumor activity of GF-P-1 depends on the activation of macrophages.

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