Clinical effect of 5-fluorouracil or chromomycin-A₃ alone, 5-fluorouracil + chromomycin-A₃, and of the first two plus prednisolone on gastrointestinal and other solid tumors was evaluated. Out of 133 cases acceptable for evaluation, the number of responders was as follows: 3 (18.8%) of 16 cases treated with 5-fluorouracil alone, 1 (9.1%) of 11 cases treated with chromomycin-A₃ or chromomycin-A₃ hemisuccinate, 13 (21.7%) of 60 cases on the two-drug regimen, and 21 (45.7%) of 46 cases on the three-drug regimen. In cases of stomach carcinoma, response rate to the three-drug regimen was 54.2% (13/24), significantly higher than that of other regimens. At least 25% regression in the size of primary tumor was observed in 2 (7.1%) of 28 cases on the two-drug regimen and in 6 (33.3%) of 18 cases on the three-drug regimen.
Of 51 cases on the three-drug regimen, steroid diabetes developed in 5 cases, moon face in 4 cases, and gastric ulcer in 1 case. However, toxic effect of these regimens (especially appearance of leucopenia) was less than those of previously tried combined regimens. The duration of response, on an average, was 10.8 weeks in 13 cases on the two-drug regimen and 11.7 weeks in 21 cases on the three-drug regimen.
It was concluded from these results that a better response is obtained by the three-drug regimen than other regimens, and that prednisolone in combiation, in addition to its favorable effect in improving the general condition of the patients, might enhance the anticancer effect of the drugs used in combination.

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Corynebacterium liquefaciens can induce a marked resistance to tumor cell (AH-7974) challenge in Donryu rats. This effect was also induced in T-cell-deficient athymic nude mice. The macrophages harvested from the peritoneal cavity of a rat injected with C. liquefaciens showed a marked inhibition of tumor growth in vitro. Therefore, the effector cells were assumed to be peritoneal macrophages. The time needed for activation of macrophages in vivo was within 3 to 5 days after injection of C. liquefaciens. Before or after this period, the peritoneal macrophages did not inhibit tumor cell proliferation. Furthermore, normal peritoneal macrophages were activated in vitro by C. liquefaciens and displayed remarkable antitumor activity. These results suggest that C. liquefaciens rendered the macrophages inhibitory to tumor cells even under T-cell depleted condition. On the other hand, in vitro antitumor activity of the activated macrophages disappeared completely after in vivo treatment of the peritoneal cavity of a rat with Trypan Blue, a lysosomal enzyme blocker. Resistance of the rat induced by C. liquefaciens to tumor cells was also abrogated by Trypan Blue treatment just 1 day before tumor cell inoculation. Trypan Blue blocked the acid-phosphatase (a marker enzyme of lysosomes) activity of the activated macrophages but it seemed not to affect other macrophage activities, at least in the following two points; normal macrophages treated with Trypan Blue had almost normal phagocytic function of India ink particles and, moreover, they could be rendered tumoricidal, same as normal macrophages, in vitro by C. liquefaciens.

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The effect of the nephrotoxic substance, N-(3, 5-dichlorophenyl) succinimide, on the histological pattern and incidence of kidney tumors induced by streptozotocin in rats was studied. In groups administered streptozotocin alone or in combination with nicotinamide, the histological pattern and the incidence of kidney tumors in rats were similar; renal cell tumors developed in 1 of 9 rats (11.1%) and in 2 of 17 rats (11.8%), respectively. However, post-treatment with N-(3, 5-dichlorophenyl) succinimide increased the induction of epithelial tumors by streptozotocin, and embryonal cell tumors were also induced nearly as frequently. Administration of nicotinamide alone did not result in the development of kidney tumors.

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Glucocorticoid hormones induced differentiation of mouse myeloid leukemia cells. From comparison of the structure of steroids with their ability to induce phagocytic and locomotive activities, typical characters of mature macrophages and granulocytes, the simplest steroid with inducing ability was concluded to be a steroid with the structure of progesterone and one hydroxyl group at 11β- or 21-position. The maximum induction ability seemed to require the structure of progesterone and three hydroxyl groups (at 11β-, 17α-, and 21-positions). A single, 30-min pulse treatment with glucocorticoid was sufficient to induce differentiation of leukemia cells.
Glucocorticoid receptors were detected in mouse myeloid leukemia cells. The binding affinity of various steroids for the cytoplasmic receptors was closely correlated with the activities of these compounds to induce differentiation of leukemia cells, suggesting that these receptors may be involved in hormonal induction of differentiation of various cells. This suggests that the binding reaction is important for differentiation of myeloid leukemia cells.

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Sera from patients with gastric carcinoma markedly suppressed the normal lymphocyte response to phytohemagglutinin (PHA) when compared with normal sera. The effect of this inhibition was well correlated with the degree of advance of the disease.
Fractionation of normal and cancerous sera was carried out by DEAE-cellulose column chromatography using a continuous concave gradient. The elution patterns of the two kinds of sera were quite similar and separated into nine fractions. Protein analysis by immunoelectrophoresis and immunodiffusion showed no difference in subfractions between normal and cancerous sera. However, inhibitory activities of cancerous sera on PHA response of normal lymphocytes were found in α-globulin, β-globulin, and α₂-macroglobulin fractions. On the other hand, in the fractions from normal serum, α-and β-globulin fractions showed a similar degree of inhibition to PHA response of normal lymphocytes, but α₂-macroglobulin fraction had no inhibitory activity. Such inhibitory fractions showed an immunosuppressive activity not only to PHA response of normal lymphocytes in vitro, but also to the induction of splenic plaque-forming cells in vivo. The electrofocusing patterns of α₂-macroglobulin fractions obtained from normal and cancerous sera showed a remarkable difference in protein composition.
These results indicated that the function of lymphocytes may be regulated by a variety of humoral factors and, especially in patients with gastric carcinoma, the cell-mediated immunity may be modified by some factors not present in normal serum.

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The possibility of cloning avian tumor virus (ATV) by the transfection technique was studied. The attempts required efficient facilitator for the uptake of exogenous cellular DNA by chick embryo cells (CEC) to increase transfection efficiency. The calcium method was more efficient than the DEAE-dextran method for the uptake of tritium-labeled DNA, and the calcium method was used in further segregation studies of ATV by transfection of cellular DNA from ATV-infected cells. The test viruses included the BAI-A strain of avian myeloblastosis virus (a mixture of subgroup A and B viruses) and the B77 strain of avian sarcoma virus (a mixture of transforming and transformation-defective viruses). Analysis of the recovered progeny viruses after transfection revealed that they were indeed segregates of the parent viruses.

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Serum β₂-microglobulin levels were measured by radioimmunoassay in patients with various malignant neoplasms, ascitic patients, and also patients with definite or suspected hepatoma showing variable levels of serum α-fetoprotein. Elevated serum β₂-microglobulin levels greater than 2.5mg/liter were found in various malignant neoplasms, especially in multiple myeloma (66.6%) and hepatoma (60.4%). The ascites/serum ratio of β₂-microglobulin levels in the patients with malignant ascites is significantly higher than in those with non-malignant ascites. However, ascites/serum ratios of total protein, IgG, albumin, and creatinine levels were not significantly different between the two groups. Levels of serum β₂-microglobulin were correlated well with those of α-fetoprotein in the patients with definite or suspected hepatoma (γ=0.72, P<0.001).
From these results it was concluded that (1) high levels of serum β₂-microglobulin in these patients could be attributed to its hyperproduction by tumor cells or by the cells which had been infiltrated and activated, (2) it is useful to estimate the ascites/serum ratio of β₂-microglobulin levels in differentiating malignant from non-malignant ascites, and (3) it might suggest that a function of β₂-microglobulin is in some way related to that of α-fetoprotein, and the α-fetoprotein-synthesizing cells secrete a great deal of β₂-microglobulin, although its function remains unclear.

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Conditioned medium containing a differentiation-stimulating factor (D-factor) for M1 cells (myeloid leukemia cell line) was recovered over a period of several weeks by culturing mouse secondary embryo cells in serum-free medium but supplemented with bovine serum albumin. Incubation of M1 cells with the conditioned medium for 48hr or less resulted in differentiation of 60 to 80% of the cells. From the conditioned medium, D-factor was partially purified by preparative electrophoresis on a Pevikon block and the specific activity was increased 50∼60-fold. The partially purified D-factor was capable of differentiating 30% of the M1 cells at a protein concentration of 2∼3μg/ml. Half-logarithmic plots of phagocytosis-inducing activity vs. protein concentration yielded a sigmoid dose-response curve. D-factor is considered to be a glycoprotein with a molecular weight of 40, 000∼50, 000 and an electrophoretic mobility of α-globulin. It is resistant to different protein-denaturing treatments. Since the nature of D-factor differs from that of the colony-stimulating factor for mouse bone marrow cells, it appears that these two activities are due to different substances.

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Reactivity of lymphocytes from 115 gastric cancers to allogeneic tumor antigens and to myelin basic protein was studied by the macrophage electrophoretic mobility test, and its result was compared with lymphocyte reaction to autochthonous tumor antigens. Lymphocytes from gastric cancer showed a positive response in 72.2% of reactions to allogeneic tumor extracts from gastric cancer, and in 74.5% of those from gastric sarcoma. The incidence of positive reaction to allogeneic antigens was slightly lower than that to autochthonous tumor antigens. Lymphocytes from breast and lung cancers also reacted with tumor extracts obtained from gastroduodenal malignancies. These results suggest the presence of a common tumor antigen which is distributed among various types of malignancies and stimulates lymphocytes only from cancer patients. Combination of lymphocytes and antigens both from high responders gave positive reaction in 73.9% of examinations. Positive reactions were also observed in 61.4% of a combination of lymphocytes from high responders and antigens from low responders, in 23.6% of a combination of lymphocytes from low responders and antigens from high responders, and in 14.9% of a combination of lymphocytes and antigens both from low responders. These results indicate that lymphocyte reactivity is more relevant to the level of macrophage electrophoretic mobility reaction than antigenic potential of the tumor extracts. Some of allogeneic antigens showed a correlation with autochthonous antigens in the pattern of macrophage inhibition, and others did not so well. Myelin basic protein was also in good correlation with autochthonous antigens, which suggests the possibility of its serving as a standard antigen for this test.

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Combination effect of antitumor agents, including doxorubicin and daunorubicin, was evaluated on the concept of pharmacological synergism in ascites sarcoma-180 system. In alternate administration, combinations of doxorubicin plus cyclophosphamide, thio-TEPA, carboquone, actinomycin-D, vinblastine, vincristine, methotrexate, cytarabine, 6-mercaptopurine, or L-asparaginase showed synergism, but in simultaneous one, only three agents, cyclophosphamide, carboquone, and cytarabine, were synergistic. On the other hand, combination of daunorubicin plus one of 8 agents (thio-TEPA, mitomycin-C, bleomycin, actinomycin-D, vinblastine, ancytabine, 6-mercaptopurine, and L-asparaginase) and 6 agents (cyclophosphamide, thio-TEPA, mitomycin-C, bleomycin, actinomycin-D, and vinblastine) provided synergism in alternate and simultaneous administration. Combination effect of agents was affected by the schedule of drug administration for doxorubicin, but weak for daunorubicin. Toxicity of doxorubicin or daunorubicin in combination with other drugs was also affected by the schedule of administration. Combination of a larger number of agents in simultaneous administration provided antagonism compared with an alternate administration.

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Bacterial growth within 72hr after an intravenous inoculation with Listeria monocytogenes was enhanced strikingly in the liver of mice, when viable cells of sarcoma-180 were injected subcutaneously into ddN, C3H/He, and BALB/c mice 5hr before the inoculation. Such an enhanced bacterial growth appears to be attributable to a depressed ability of macrophages to digest engulfed bacteria. Pretreatments with zymosan, killed BCG, or viable BCG prevented such depression in tumor-bearing mice and increased the bactericidal activity in the liver of normal and tumor-bearing mice above the level of non-treated normal mice. Such adjuvants may be useful not only for augmentation of antitumor activity but also for augmentation of antimicrobial activity in tumor-bearing hosts.

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The carcinogenicity of 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) was investigated by administering it orally to Sprague-Dawley female weanling rats in a grain diet at a dose of 0.2% for 46 weeks, followed by 20 weeks of unmedicated control diet. The total mean cumulative dose of AF-2 was 10.1g (41.6mmol) per rat during the 46 weeks of the experiment. Weight gain of rats fed AF-2 was comparable to that of unmedicated control rats. Survival of rats fed AF-2 was 52±12 (SD) weeks compared to 65±5 weeks for the unmedicated controls (P<0.001). Twenty-four of 29 rats fed AF-2 developed multiple breast tumors, 15 of which were adenocarcinomas. Two of 29 unmedicated control rats had single fibroadenomas of the breast.

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Cytotoxicity of lymphocytes against allogeneic target cells of a bronchogenic squamous cell carcinoma was examined by a microcytotoxicity test in lung cancer patients after various kinds of anticancer treatment. Cytotoxicity in untreated cases at stages I, II, and III was higher than the range of controls, but cytotoxicity in those at stage IV decreased to the range of controls. Tumor resection augmented cytotoxicity in patients at stages II and III, when the assays were carried out 3 weeks after the operation. Local irradiation of tumors augmented cytotoxicity in patients a tstages II, III, and IV, when the assays were carried out 1 week after the last irradiation. Immunotherapy with BCG cell-wall skeleton augmented cytotoxicity to a marked degree in patients at stages I, II, III, and IV, when the assays were carried out after 4 months of a continuous treatment. The microcytotoxicity test may be useful for estimating the reactivity of lymphocytes in lung cancer patients in various situations.

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Serial evaluation of host immunocompetence was made by the phytohemagglutinin (PHA) skin test in 13 patients with leukemia and malignant lymphoma, receiving antineoplastic chemotherapy to induce remission.
A rebound phenomenon of skin reactivity to PHA was observed after each period of chemotherapeutic treatment in patients with a good response to therapy, but not in patients with a poor prognosis. It is concluded that sequential evaluation of skin reactivity to PHA, as well as in vitro lymphocyte stimulation, is valuable clinically in assessing the progress of malignancies and the response of patients to therapy.

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Groups 1, 2, and 3 of female Donryu rats were given continuously 400, 200, or 100ppm solution of 1-methyl-1-nitrosourea (MNU) as their drinking water. The incidence of neurogenic tumors was 12/27 (44%), 30/33 (91%), and 33/36 (92%) in Groups 1, 2, and 3, respectively. Among the neurogenic tumors, neurinomas developing from the spinal nerve roots were the most frequent. In addition, tumors of the digestive tract were found in 12, 1, and 2 rats in Groups 1, 2, and 3, respectively, and tumors in hematopoietic tissues developed in 6 rats. Tumors in other organs were infrequent.

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The rapid induction of hyperplastic liver nodules by treatment with diethylnitrosamine (DEN) followed by administration of several kinds of chemicals and hepatectomy was studied in male Sprague-Dawley rats. Rats were injected with 200mg/kg body weight of DEN intraperitoneally and 2 weeks later a second chemical (N-2-fluorenylacetamide (2-FAA), 3'-methyl-4-(dimethylamino) azobenzene (3'-Me-DAB), dimethylnitrosamine (DMN), DEN, DL-ethionine, quinoline, 5, 7-dibromo-8-hydroxyquinoline, 8-hydroxyquinoline, or 8-nitroquinoline) was administered for 2 weeks, and partial hepatectomy was performed at the end of the third week of the experiment. Significant difference in the number of hyperplastic nodules was found in groups treated with 2-FAA, 3'-Me-DAB, DEN, DMN, DL-ethinonine, or quinoline compared with the control group. In groups treated with the same experimental design but without partial hepatectomy, no significant difference in the number of hyperplastic nodules was found. Continuous administration of each of the above chemicals for 4 weeks, with or without partial hepatectomy, also did not induce a significant number of hyperplastic nodules.
Histochemically, γ-glutamyltranspeptidase (γ-GTPase) (EC 2.3.2.1) was demonstrated in hyperplastic nodules but was absent in non-hyperplastic areas. The activities of acid phosphatase (EC 3.1.3.1), glucose-6-phosphatase (EC 3.1.3.9), and adenosine triphosphatase (ATPase) (EC 3.6.1.3) were demonstrated in non-hyperplastic areas but were low or deficient in hyperplastic nodules. γ-GTPase, as positive reaction, and ATPase, as negative reaction, are particularly good markers for the detection of hyperplastic nodules. The present results indicate the possible usefulness of a system consisting of 3 components as an in vivo short-term screening test for hepatocarcinogens: (1) DEN injection, (2) test chemical administration, and (3) partial hepatectomy during chemical administration.

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Spleen cells from WKA rats immunized with allogeneic Donryu tumor (AH-66), when examined by the Winn test, inhibited lethal growth of admixed antigenically unrelated syngeneic tumor (KMT-17) only when a mixture of spleen cells and tumor cells was injected into pre-irradiated recipients. On the other hand, spleen cells specifically sensitized to KMT-17 inhibited growth of admixed KMT-17 cells either in irradiated or non-irradiated rats.

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Cell-free ascites from syngeneic MM46 tumor-bearing C3H/He mice inhibited the antibody-dependent macrophage-mediated tumor lysis in vitro and the inhibitory activity increased with the period of tumor bearing. Thus, the inhibitory activity was demonstrated at the effector cell level. Three fractions of lipoprotein and a protein-rich fraction were prepared from the cell-free ascites by sequential flotation. Among these, the very low density (ρ<1.006) and low density (1.006<ρ<1.063) lipoprotein fractions inhibited tumor lysis mediated by activated macrophages and syngeneic antitumor antibody, whereas the high density (1.063<ρ<1.21) lipoprotein and the protein-rich infranatant fractions did not. This inhibitory activity at the effector cell level may be due to functional depression of macrophages by lipoprotein fractions.

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The carcinogenic activity of young bracken dried at a high temperature or minced by a mechanical chopper was compared with that of unprocessed bracken dried at room temperature. Inbred strain ACI rats of both sexes were divided into 4 groups; Group 1 received a diet containing the unprocessed bracken dried at a room temperature of below 30°, Group 2 received a diet containing bracken dried at 70∼90° with a hot, forced draft, and Group 3 received the diet containing processed bracken that had been minced to a paste-like consistency using a mechanical chopper. The control group was fed a normal diet. Rats fed a bracken-containing diet developed tumors most frequently in the ileum. Comparison of Group 1 with Groups 2 and 3 showed no significant difference in the latent period, incidence, histologic types, or multiplicity of the intestinal tumors found in each animal.

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A rapid, reproducible method for the production of ulcers and regenerative hyperplasia of the urinary bladder of rats is described. Ulceration was produced by freezing the serosal surface of the bladder. At 5 days the regenerative hyperplasia became marked showing papillary and nodular formation.

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An ovary-dependent mammary tumor was successfully isolated from BALB/c mice after oral administration of urethan. The tumor line was characterized by apparent growth in intact female mice and insignificant growth in ovariectomized or testectomized mice.

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