The acetone-dried powder, obtained from the mother liquor left after separation of the pH 5.4-precipitating fraction (crude Parotin) from the aqueous extract of a bovine parotid gland, was extracted with glacial acetic acid and its product was purified to a protein (mol. wt. 66000) showing homogeneity in disc electrophoresis. Since this substance was expected to have an antitumor activity, because of its action to increase the immune competence besides lowering serum calcium in rabbits, its effect on a solid tumor was examined with sarcoma-180 transplanted subcutaneously in Swiss-Webster or ICR-SLC strain mice. Four-weekold mice were divided into two groups; one group was given the test solution and the other injected with saline as a control. Subcutaneous injection of 20μg/ mouse (corresponding to about 1mg/kg) of the purified product 1 week before transplantation of sarcoma-180 in Swiss-Webster strain mice resulted in complete regression of the tumor in all of 8 mice, while subcutaneous injection of 1μg/mouse (corresponding to about 0.05mg/kg) of the product every day for 10 days after the transplantation also showed a tumor inhibition rate of 97.9%, the tumor disappearing in 5 of 6 mice. Intraperitoneal injection of 1μg/mouse of the product every day for 10 days after the transplantation showed a tumor inhibition rate of 96.0%, the tumor disappearing in 3 of 6 mice.
Examination of the thymus gland, liver, spleen, and lymph nodes at the time of autopsy 35 days after transplantation of sarcoma-180 showed, on the whole, no special changes in these organs, except for greater swelling of the inguinal lymph node on the transplanted side when growth of the tumor was marked.

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Human gastric and colon carcinomas serially transplantable in nude mice, designated as St-4, St-15, and Co-3, were used for chemotherapeutic experiments. All tumors showed a 100% take rate and stable growth.
Mitomycin-C (MMC), 3mg/kg body weight, given intraperitoneally 4 times once weekly, showed some inhibiting effect on Co-3, St-15, and St-4 in that order. N¹-(2'-Tetrahydrofuryl)-5-fluorouracil (FT-207), 90mg/kg body weight, intraperitoneally daily for 4 weeks, suppressed the growth of Co-3 and St-15 slightly but with statistical significance, and had no effect on St-4. Co-3, a tumor with more rapid growth and richer vascular supply, was suppressed more by MMC and FT-207.
These three transplantable tumors may be useful, not only in evaluating chemotherapeutic agents for use against human gastrointestinal carcinomas, but also for screening of new antitumor agents.

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Oil-attached BCG cell-wall skeleton (BCG-CWS) was demonstrated to have an activity inducing lymphocyte trapping in the draining node in rats. It acts also as a potent adjuvant for the lymphocyte trapping when injected into the growing syngeneic transplantable tumor.
Treatment with repeated intratumor injections into the primary tumor resulted in suppression of tumor growth in both primary and metastatic sites. Even when the primary tumor escaped regression, inhibitory effect on metastatic spread was attained by the therapy.
The contribution of BCG-CWS to suppression of metastasis especially in the draining node was discussed from the point of lymphocyte trapping in the draining node.

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Changes of the cell cycle during carcinogenesis of hamster cheek pouch epithelium were studied and it was demonstrated that in hyperplastic and neoplastic stages, the cell cycle time decreased to approximately 2/3 and 1/3 of the normal control, respectively. A nominal growth fraction was found to exceed unity in carcinogen-treated epithelium, whether hyperplastic or neoplastic. This result seems to indicate that a considerable number of cells in which DNA synthesis is stimulated by the carcinogen fail to enter mitosis. A possible implication of repair synthesis is also discussed.

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The relationship between tumor resistance and the antitumor delayed hypersensitivity reaction (TDHR) after resection of the tumor was studied by using the syngeneic 3-methylcholanthrene-induced murine tumor, MCA-DDD. MCA-DDD was inoculated subcutaneously into the tail of DDD mice and 4 weeks later the tumor-bearing tail was resected. The tumor resistances of the mice were then determined from the diameters of the tumor that developed 14 days after subcutaneous challenge of the mice with tumor cells in the flank.
It was found that the mice showed a specific resistance to the tumor until 30 days after tumor resection. In parallel with tests on tumor resistance, TDHR of the mice after tumor resection was examined by the footpad test. The cell-free fraction of sonicated MCA-DDD tumor cells was used as the preparation of tumor antigens. TDHR of the mice appeared soon after tumor resection, reached a maximum on day 7, and then decreased slowly until day 29.
Mice that acquired tumor resistance showed rapid increase of TDHR after challenge with fresh tumor cells. TDHR was high on day 4 after the challenge and its level was well correlated with tumor resistance of the mice.

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Humoral and cellular immune responses were investigated after combination therapy with syngeneic antitumor serum and bacterial lipopolysaccharide (LPS). The titer of antitumor antibody determined by using a macrophagemediated system was very high in mice cured by the combination therapy, and this high titer lasted for a long time. In contrast, no significant titer was detected using an antibody-dependent lymphocyte-mediated system. Thus, antibody-dependent macrophage-mediated cytolysis was a more sensitive method for detecting antitumor antibody. The cellular immune response was measured as the delayed hypersensitivity reaction to tumor cells. In mice that had been cured by combination therapy, this reaction appeared at an early stage, before any antitumor antibody was detectable, but it soon decreased. On the other hand, tumor-bearing mice showed a low level of antibody and no significant delayed hypersensitivity reaction.

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Three phenylimidazothiazole isomers, L-(-)-form (levamisole), D- (+) -form (dexamisole), and DL- (±) -form (tetramisole), modulate mouse and rat immune responsiveness. They depress the relapse of urinary bladder cancer, BC-47, when they are used with 1, 3-bis (2-chloroethyl)-1-nitrosourea (BCNU) for treatment of rats bearing BC-47. Levamisole was the most effective isomer. Effectiveness of the isomers is expressed by their ability to enhance the number of plaque-forming cells to sheep red blood cells. They also stimulate the DNA synthesis of murine lymphocytes in vitro.

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Immunopotentiated rats, which were injected with Propionibacterium acnes or BCG, had the 50% survival twice as long as those in untreated controls after intravenous inoculation of Sato lung carcinoma (SLC) cells. The amount of labeled tumor cells in the lung of the adjuvant-treated rats decreased significantly in the first 20hr after intravenous injection of ⁵¹Cr-labeled tumor cells compared to that of control animals. The elevated activities of ATPase and acid phosphatase in the whole nucleated spleen cells as well as spleen lymphocytes separated by Ficoll-Conray gradient were also demonstrated in adjuvant-treated groups. These data suggested that the elevation of ATPase and acid phosphatase activities in nucleated spleen cells as well as spleen lymphocytes has an important role for the suppression of tumor growth in adjuvant-treated rats.

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Tumor induction by fractionated whole-body X-irradiation (400 rad) was studied in spayed Sprague-Dawley rats. Ovariectomy was chosen as an intensifying factor for radiation leukemogenesis. Ovariectomized rats gained more body weight and responded more quickly (but transiently) in the recovery of WBC levels after the last (3rd or 4th) X-irradiation. A total of 26 tumors developed in 21 out of 47 ovariectomized rats, 11 tumors in 6 out of 13 ovariectomized and ovary-grafted rats, and 44 tumors in 25 out of 29 sham-ovariectomized rats during the observation period up to 64 weeks after starting X-irradiation. Eighty per cent of tumors were of mammary gland origin in the latter two groups with intact or grafted ovaries. By contrast, 61.1% of tumors in the spayed rats were derived from the subcutaneous mesenchymal tissue and the hematopoietic tissue. This may imply that some forms of mesenchymal tumors including leukemia are under the suppressive influence of female sex hormones.

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Differences in susceptibility to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) of fundic mucosa in various states of regeneration after induction of ulcer with iodoacetamide were examined histologically in male Wistar rats. Iodoacetamide was given to rats in their drinking water, before (Group 1), with (Group 2), or after (Group 3) MNNG. Atypical hyperplasia in the renewed mucosa and pyloric gland metaplasia were observed on the ulcers in Group 1 in higher incidence than in Groups 2 and 3. In addition, adenocarcinoma developed in the ulcer of 2 of 17 effective animals in Group 1. These observations suggest that the mucosa showing pyloric gland metaplasia is more susceptible to MNNG than the young rapidly regenerating mucosa at the margin of ulcers.

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As the prototypes for proximate and ultimate metabolite of hepatocarcinogenic N-methyl-4-aminoazobenzene (MAB) dyes, N-hydroxy and N-acyloxy derivatives were synthesized from MAB, 3'-methyl-MAB, and 4'-methyl-MAB, whose carcinogenic activities in the rat are known to be quite different, and electrophilic reactivity and mutagenicity of the parent dyes and the N-hydroxy and N-acyloxy derivatives were comparatively studied. MAB and its ring-methyl derivatives did not show reactivity with amino acids, proteins, or with DNA, and N-hydroxy derivatives reacted a little except with cysteine. Mutagenicity of these dyes towards Salmonella typhimurium TA-98 and TA-100 was expressed only in the presence of 9000g supernatant of a rat liver homogenate (S-9 Mix), and either 3'-methyl or 4'-methyl substituent did not affect the mutagenic activity of the parent dyes. By contrast, N-acetoxy-MAB, N-benzoyloxy-MAB, and their ring-methyl derivatives showed a high degree of reactivity with amino acids, proteins, and DNA, and they were strongly mutagenic towards the bacteria without S-9 Mix treatment. No critical differences were observed among these N-acyloxy derivatives either in their electrophilic reactivity or mutagenicity.
These findings suggest that neither the rate of metabolic activation by liver enzymes from MAB homologs to the ultimate metabolite nor electrophilic or mutagenic activity of the prototype compounds for the ultimate metabolite is influenced by the ring-methyl substituent which affects the carcinogenic activity of the parent dyes. Other possible factors influencing the carcinogenic activity of MAB homologs are discussed.

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Examinations were made on substances that enhance or inhibit the induction of hepatoma in rats previously fed 3'-methyl-4-(dimethylamino) azobenzene (3'-Me-DAB) for a brief period. The substances tested were stilbene, 4-nitrostilbene, 4, 4'-dihydroxystilbene, diethylstilbestrol, 17β-estradiol, and methyltestosterone. Male Donryu rats were fed 0.5g of 3'-Me-DAB by being maintained on a diet containing 0.06% 3'-Me-DAB, and then they were fed 0.25 or 0.5g of a test substance with the basal diet. Comparison of the development and yield of hepatomas indicated that 4-nitrostilbene and methyltestosterone had an activity of enhancing 3'-Me-DAB carcinogenesis, whereas diethylstilbestrol and 17β-estradiol had an activity to retard it. Other substances showed no such activities.
The enhancement by 4-nitrostilbene and inhibition by diethylstilbestrol of 3'-Me-DAB carcinogenesis was correlated with their effect on liver nucleic acid metabolism. Feeding of 4-nitrostilbene caused a selective inhibition of Mn²⁺-(NH₄)₂SO₄-activated RNA polymerase activity of liver nuclei and reduced liver RNA content. The deleterious alteration of liver RNA metabolism was followed by the enhancement in the incorporation of ip-injected ³H-thymidine into DNA of liver nuclei. On the other hand, feeding of diethylstilbestrol increased tissue RNA content without effect on RNA polymerase activity of liver nuclei, and had an activity of increasing the incorporation of ³H-thymidine into DNA. The possible implication of these results with regard to the enhancement and inhibition of hepatocarcinogenesis is discussed.

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Carcinogenicity of an aqueous extract of bracken was studied in inbred strain ACI rats. In Experiment 1, Group 1 rats received a diet containing unprocessed dry bracken powder for 3 months, Group 2 received the boiling water extract of dry bracken as drinking water for 16 months, Group 3 received the cold water extract of dry bracken as drinking water for 16 months, and Group 4 received a diet containing the concentrated boiling water extract of dry bracken during the whole experimental period. Rats in Experiment 2 also received a diet containing the boiling water extract of dry bracken. A control group was fed a normal diet. Majority of the animals in Group 1 developed multiple ileal tumors, such as adenomas and adenocarcinomas. In Group 2, 6 of 11 rats which survived beyond 7 months developed tumors in either the ileum or urinary bladder, or in both. Rats in Group 3 had no tumor. In Group 4 and in Experiment 2, all the animals except 1 rat in Experiment 2 had urinary bladder tumor. Furthermore, almost all the rats in these groups had simultaneously ileal tumors. Based on these results, it may be assumed that the carcinogen in bracken is extractable in boiling water and is most probably also water-soluble.

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The stationary phase of cell growth in suspension culture was analyzed with regard to nutritional depletion using FM3A cells originally derived from a spontaneous mammary carcinoma in a C3H mouse. When cells reached the stationary phase at the cell density of 5×10⁵ cells/ml, cells began to lose the ability to form colonies and viability as determined by the dye exclusion test. When the amount of amino acids and vitamins in the medium decreased, cells reached the stationary phase at a low terminal density. Cells in the spent medium supplemented with amino acids, vitamins, glucose, and serum grew with the same growth rate as in the fresh medium. It is concluded that depletion of nutrients, especially amino acids and glucose, is the reason for the establishment of the stationary phase.

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The effect of phenacetin and caffeine on urinary bladder carcinogenesis in rats treated with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) was examined. Animals were given a high or low dose of BBN for 4 weeks and then phenacetin or caffeine was administered for 30 or 32 weeks, respectively. All the animals were examined histologically after 36 experimental weeks.
The incidence of papillary or nodular hyperplasia, papilloma, and cancer of the urinary bladder was significantly higher in the groups treated with BBN and then with phenacetin than in those treated with BBN alone, especially with a high dose of BBN. Simple hyperplasia and papillary or nodular hyperplasia developed in the urinary bladder of rats treated with phenacetin alone. Papillary proliferative growth of the renal pelvis was seen in one rat treated with a low dose of BBN and phenacetin.
Treatment with caffeine after BBN had no enhancing effect and caffeine alone caused no remarkable changes.

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To indicate the influence of operative stress on tumor growth, thoracotomy and/or laparotomy were performed 48hr after intraperitoneal or intravenous inoculation of Sato lung cancer into Donryu rats. Survival period, number of metastatic nodules on the surface of the lungs, and the percentage-area of metastases in the frontal section through pulmonary hilus were examined. By thoracotomy and laparothoracotomy the survival period of the tumor-inoculated rats was reduced significantly compared with that of the control but difference between these two test groups was not significant. Also there was no significant difference between the laparotomy group and the control. The results obtained in the number and percentage-area of metastatic nodules were quite similar to that observed in the survival period. Correlation between the number and the percentage-area of metastatic nodules was highly significant. The meaning of the stress of thoracotomy in cancer treatment is discussed.

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Neocarzinostatin, an antineoplastic agent which is effective against human leukemia, induced unscheduled DNA synthesis in human leukemic leucocytes. This indicated the existence of repair process against at least a part of DNA damage caused by the agent. The extent of unscheduled DNA synthesis increased in parallel with the concentration of Neocarzinostatin up to 5μg/ml, followed by a decline at more than 5μg/ml. Leucocytes from patients with chronic myelogenous leukemia had higher repair synthesis after Neocarzinostatin treatment compared to those from patients with acute leukemia. The amount of repair synthesis correlated well with the proportion of immature leucocytes among chronic myelogenous leukemia samples, but such correlation was not found among acute leukemia samples.

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Mason-Pfizer monkey virus (M-PMV), originally isolated from mammary carcinoma of a rhesus monkey, is infectious to various human cells in tissue culture. Human epithelioid cells (HeLa and HEp-2), fibroblastic cells (embryonic cells as well as virus-transformed embryonic RSb cells), and glial cells (KC) have all been infected with M-PMV. Electron microscope studies have shown that all infected cells produce progeny virus. The titer of infectious M-PMV in cultures of HeLa and HEp-2 cells assayed by KC syncytium formation was 10¹ syncytium-forming unit (SFU) per 0.2ml. The titers for whole human embryonic cells (HEC), human embryonic lung cells (HEL), and embryonic skin Detroit-551 cells were 10⁴ SFU/0.2ml for each, and 10³ SFU/0.2ml for RSb and KC cells.

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To measure immunological reactivity or recognition of tumor antigens, mixed lymphocyte-tumor cell culture reaction (MLTR) was studied using a micromethod. The peripheral lymphocytes of cancer patients were mixed in vitro with autochthonous tumor cells that had been irradiated with ⁶⁰Co at a dose of 6, 000 rad just before use. The lymphocyte blastogenic response was estimated by the incorporation of ³H-thymidine in the acid-insoluble fraction and expressed as counts per minute. The stimulation index equal to or greater than 2.0 was arbitrarily expressed as positive blastogenic response in the present experiment. Positive blastogenic responses to autochthonous tumor cells were observed in 10 of 34 patients with various solid tumors. The intensity of blastogenic response to tumor cells was correlated with that of the non-specific responses to PHA (r=0.4732, P<0.01). These results indicated that non-self tumor-associatedantigens, which could induce in vitro blastogenic response of autologous lymphocytes, were present in some part of human tumors.

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Studies were made on the effect of 0.06% α-benzene hexachloride (α-BHC) on hepatic changes, including development of hepatomas, in rats treated with 0.025% 2-fluorenylacetamide (2-FAA) at the same time.
α-BHC inhibited the induction of hepatocellular carcinomas and oval cell infiltration in rats treated with 2-FAA for 5 months. However, it did not inhibit induction of hyperplastic nodules, it induced liver adenocarcinomas in 4 of 11 rats, and it caused remarkable cyst formation in the liver of rats treated with 2-FAA.

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Tumor-specific and skin-reactive antigen of a syngeneic liposarcoma (H-10) of Hartley/F guinea pig was solubilized with 3M potassium chloride and purified by precipitation with 2M ammonium sulfate, followed by Sephadex G-200 gel filtration. The antigenic activity of 7 fractions obtained was estimated by the delayed-type skin reaction elicited in syngeneic animals immunized with H-10 cells admixed with BCG. Accurate relative activity of the fractions comparable to the skin reaction elicited by living H-10 cells was calculated by the parallel line assay method in which the dose-response curves of the fractions are compared with that of living cells. About 30∼50μg protein of the 3 fractions eluted slowly from the Sephadex column elicited the skin reaction equivalent to that elicited with 1×10⁶ of living H-10 cells. Tumor-specific skin reactivity per μg protein of these 3 fractions was roughly 20∼40 times higher than that of lyophilized cells.

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Chemotherapy of methylnitrosourea-induced autochthonous large bowel cancer in rats, which is similar to that in man, was studied to evaluate the intrarectal administration or topical application of chemotherapeutic agents. Rats with large bowel tumors confirmed by endoscopic examination received an intrarectal instillation of 1mg of 1- (4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), 1mg of 1-(2-chloroethyl)-3-(4-methylcyclohexyl) -1-nitrosourea (Me-CCNU), or 5mg of 5-fluorouracil (ir groups), or intraperitoneal injection of 2.5mg of 5-fluorouracil (ip group) daily for 8 weeks. All the rats, including non-treated control rats, were necropsied after the treatment. The number of large bowel tumors per rat detected by endoscopy before the treatment was mostly the same among groups, whereas that observed at necropsy after the treatment was significantly smaller in ir groups, compared to non-treated group and ip group. The tumors increased significantly in rats of non-treated group and ip group between the time of endoscopy and necropsy, but not in rats of ir groups. These results showed that the maximum tolerated dosage of the agents administered intrarectally suppressed the development of new tumors after start of the treatment and also the growth of tumors which were detected by endoscopy before the treatment.

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Separation and identification of 3-methylcholanthrene and its synthesized derivatives were examined by gas chromatography-mass spectrometry (GC-MS) system. Hydroxyl derivatives were silylated and all chemicals were applied to columns of OV-1 and Dexsil-300 at a constant temperature of 260° and 290°, respectively. Mass spectra of all derivatives were simple at ionization voltage of 20eV and their molecular ions gave base peaks in most cases. Mass fragmentography plotted against molecular ions satisfactorily separated all standard derivatives so far examined.

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Indian muntjac cells were efficiently transformed by murine sarcoma virus (MSV) and avian sarcoma viruses (ASV). When colony formation of the infected cells was examined in soft agar, many colonies were formed by the ASV-infected cells but no colony was seen in the MSV-infected cells. The ASV-transformed cell clones differed among the clones in morphology, presence of inducible ASV genome, and karyotypes.

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Experimental teratoid tumor was induced in mice fetectomized at 12∼15th day of pregnancy without using any carcinogens. The induced tumor consisted of well-differentiated tissues derived from all the three germ layers.

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This is the first EBNA-proved Japanese Burkitt lymphoma in a 29-year-old male with abdominal disease. The majority of ascites lymphoma cells were positive for EBNA and histology of adbominal tumor revealed a starry sky pattern. Terminal leukemia preceded death at 8 months from the onset.

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B6WF₁ mice were irradiated with X-rays at late fetal, neonatal, or juvenile stage. Hepatocellular tumor developed with a very high incidence in mice irradiated at neonatal stage. Incidence of hepatocellular tumor was enhanced by irradiation at late fetal stage.

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