Intravenous injection of oil-attached BCG cell-wall skeleton showed potent preventive activity for induction of lung cancer by the intrabronchial instillation of chemical carcinogens (3-methylcholanthrene, 4-nitroquinoline 1-oxide) in rabbits. On the other hand, the thymectomized rabbits developed lung cancer by instillation of chemical carcinogens at an 80% incidence, compared to 47.2% in the controls.
There were no differences in the appearance of amyloidosis between thymectomized and BCG cell-wall skeleton-treated rabbits and the controls.

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Thromboplastic and fibrinolytic activities of rat ascites tumor cells avoiding any stromal elements were examined and the role of these activities in the bloodborne metastasis was discussed.
Ten lines of tumor cells showed varied thromboplastic and fibrinolytic activities. Tumor cell lines examined were classified into four groups; (1) lines AH-130, AH-62, and AH-7974 with high thromboplastic and high fibrinolytic activities, (2) lines AH-130F(N), AH-66F, and AH-7974F with low thromboplastic and low fibrinolytic activities, (3) line SLC with high thromboplastic and low fibrinolytic activities, and (4) lines AH-109A, AH-41A, and AH-41C with moderate thromboplastic and low fibrinolytic activities.
The cell lines AH-130 and AH-130F(N), as well as AH-7974 and AH-7974F, have the same origin and showed different enzymic activities. AH-130 caused more prominent thrombus formation in the pulmonary vessels of rats in the early stage of intravenous inoculation and induced more prominent decrease in the number of platelets and fibrinogen levels in peripheral blood than AH-130F(N). Also, AH-130 developed more abundant metastatic foci in the lung 72hr and 7 days after intravenous inoculation than AH-130F(N).

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The activity of adenosine deaminase (EC 3.5.4.4.) was significantly lower in lymphocytes from patients with lung cancer than in those from healthy subjects, whereas the activity of purine nucleoside phosphorylase (EC 2.4.2.1.) was significantly higher in lymphocytes from the patients than in those from normal controls. When the one activity was plotted against the other, the plots for patients with lung cancer were all outside the frame formed by the lower and higher limits of the standard deviation of the mean of normal activities of the two enzymes.
The ratio of adenosine deaminase activity to purine nucleoside phosphorylase activity was lower in patients with lung cancer than in controls. The possible effect of this ratio on the function of lymphocytes was briefly discussed.
These enzyme activities were suggested to be useful measures of the immune responsiveness of patients with lung cancer.

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Effect of aluminium chloride on the binding of 4-hydroxyaminoquinoline 1-oxide (4-HAQO) to lung and liver DNA and RNA in mice and rats was examined. DNA and RNA from mouse and rat lung and liver were preincubated with aluminium chloride in an ice bath, and examination of the binding of 4-HAQO showed that the binding to each of nucleic acids was markedly inhibited.
Subcutaneous administration of aluminium chloride to mice and rats, extraction of DNA and RNA from their lung and liver, and examination of the binding of 4-HAQO indicated that the binding of 4-HAQO with DNA and RNA in each of the organs was suppressed in mice. On the other hand, binding of 4-HAQO to lung DNA and RNA, and liver DNA was suppressed in rats but there was no effect on the binding to liver RNA.
Simultaneous subcutaneous injection of ¹⁴C-labeled 4-nitroquinoline 1-oxide (¹⁴C-4-NQO) and aluminium chloride on different sites on the back and examination of the binding of carbon-14 to nucleic acids in the lung and liver indicated that incorporation of radioactivity into lung DNA and RNA was markedly suppressed in both mice and rats. This was especially marked in the lung DNA, the binding rate being 58.2% in mice and 46.1% in rats, as compared with the control. Binding rate to liver DNA and RNA was not different from that in the control, both in mice and rats.
Effect of aluminium chloride on the damage of DNA by 4-NQO was examined by using rat lung nuclear DNA. Damage of DNA by intraperitoneal or subcutaneous administration of 4-NQO was markedly suppressed by the intraperitoneal or subcutaneous administration of aluminium chloride.

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Donryu strain male rats were administered a large amount of N-methyl-N-nitrosobenzylamine directly into the stomach through a tube. Ulcers were found to develop in the portion of the esophagus where esophageal carcinoma is known to develop. Autoradiography of the entire body of rats after an intravenous injection of N-methyl[U-³H]-N-nitrosobenzylamine showed specific incorporation of radioactivity in the esophagus. When N-methyl[U-³H]-N-nitrosobenzylamine was injected intravenously, incorporation of radioactivity in the esophagus was higher than in other parts of the alimentary canal, and remained at the same value even after 24hr. Microautoradiography of rats injected with N-methyl[U-³H]-N-nitrosobenzylamine through the tail vein showed that the silver grains were more dominant in the mucous membrane than in the muscle layer in the esophagus. These results suggest that N-methyl-N-nitrosobenzylamine accumulated specifically in the esophagus, resulting in the production of carcinoma.

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Effect of the additional treatment with coenzyme Q₁₀ on immunorestoration with Mycobacterium bovis BCG in tumor-bearing mice was investigated. Cellmediated cytotoxicity in tumor-bearing mice against alloantigenic tumor cells was determined by ⁵¹Cr release assay using spleen cells of C57BL/6N mice which had been inoculated subcutaneously with syngeneic melanoma-B16 and immunized intraperitoneally with alloantigenic mastocytoma P815-X2 cells. The cell-mediated cytotoxicity against mastocytoma P815-X2 cells was gradually depressed with the growth of melanoma-B16. The depressed, cell-mediated cytotoxicity in tumor-bearing mice recovered slightly by the treatment with BCG. The recovery effect of BCG on the depressed, cell-mediated cytotoxicity was significantly enhanced by the additional treatment with coenzyme Q₁₀. Coenzyme Q₁₀ did not have an apparent effect on the depressed, cell-mediated cytotoxicity in tumor-bearing mice. These results show that coenzyme Q₁₀ enhances the immunorestoration with BCG in tumor-bearing mice.

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4-Nitrosoquinoline 1-oxide induced malignant tumors at the subcutaneous site of injection in mice. It affected Escherichia coli to induce the so-called UV-type lesion in cellular DNA. DNA base-quinoline adducts produced by the treatment of mammalian cellular DNA with this carcinogen were proved to be identical with those obtained by the action of 4-nitroquinoline 1-oxide. Although this carcinogen was reactive enough to modify DNA chemically by itself, a different DNA modification took place in a chemical process from those obtained in the in vivo process.

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The effect of cryosurgery and surgical operation on Moloney murine sarcoma virus-induced primary tumors in the treated and untreated groups of BALB/c mice was compared in terms of tumor growth, cumulative mortality, lymphocyte-mediated cytotoxicity, proliferative response of lymphocytes, and humoral antibody formation. The results indicate that cryosurgical treatment showed both in vivo and in vitro effects; (i) tumor growth and cumulative mortality in mice treated by cryosurgery were significantly lower than those in untreated groups, and (ii) their cellular immune response was enhanced, as manifested by increase in a proliferative response and in lymphocyte-mediated cytotoxic activity against Moloney murine leukemia virus-induced lymphoma. The proliferative response of spleen cells and the cytotoxic activity of lymphocytes were not parallel; the proliferative response detected by ³H-thymidine incorporation manifested peak activity 3 days after cryosurgery, but the cytotoxic activity detected by ¹²⁵I-iododeoxyuridine-releasing tests was considerably decreased at this stage. Two weeks after cryosurgery and thereafter, however, the cytotoxic activity of this group increased to a level higher than that of untreated or surgically treated groups.
On the contrary, surgical operation abrogated the proliferative response of spleen cells. Immunofluorescence tests revealed, however, that humoral antibody formation was higher in surgically operated groups than in other groups.

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In order to acquire a fundamental knowledge for the development of better tumor-scanning agents, the in vivo incorporation pattern of three ₁₄C-labeled D-amino acids, alanine, leucine, and tryptophan, into the tumor cells and organs of animals bearing Ehrlich mouse tumor, sarcoma-180, leukemia L-1210, or Yoshida sarcoma was investigated, and compared with that of the corresponding L-forms. The radioactivity of D-amino acids tested was most highly found in tumor cells and pancreas, and the activity in tumor cells was several times higher than that of L-forms. A large portion of the radioactivity of D-forms was found in trichloroacetic acid-soluble fraction of the cells, whereas that of L-forms was mostly in protein fraction, except L-alanine. Although the mechanisms whereby the radioactivity of D-amino acids was concentrated more than that of L-forms in the tumor cells have not yet been clearly elucidated, it was concluded that γ-emitter-labeled D-amino acids themselves or their derivatives might be useful as tumor-detecting radiopharmaceuticals.

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A transplantable ACTH-secreting pituitary tumor (AtT-20) in mice was studied for its growth in relation to adrenocortical steroid. The latency of transplanted tumor in LAF-1 mice with 10⁶ tumor cells was about 2 weeks in adrenalectomized and in intact mice. In the former, however, tumor size reached 1.5cm in diameter within 1 month, while it remained about 0.5cm in the latter. Macroscopic and microscopic observation revealed that the tumor grafted in intact mice showed massive necrosis, leaving minimum intact tumor cells among necrotic foci. In contrast, the tumor grafted in adrenalectomized mice was healthy, soft, and showed adenomatous structure. It is concluded that excess of adrenocortical steroid produced in the grafted AtT-20 was inhibitory for the growth of transplanted tumor and removal of the adrenal glands was good for growth and survival of transplanted tumor. For the assessment of direct effect of adrenal steroid on AtT-20 cells, incorporation of ³H-thymidine in cultured AtT-20 cells was examined in a medium supplemented with glucocorticoid hormone. The autoradiographic study indicated that the inhibition of ³H-thymidine uptake was demonstrated at 24hr after incubation with steroid and labeled cells were barely detectable on 3rd and 5th day.

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Covalent binding of 2-acetaminofluorene[9-¹⁴C] with exogenous Torula yeast RNA and endogenous protein was investigated in liver microsome system in vitro. The binding to protein was 100 times higher than that to RNA. Requirement of NADPH, effectiveness of methylcholanthrene treatment, and inhibition by 7, 8-benzoflavone suggest possible involvement of mixed-function oxidases in this binding. The binding was not due to contaminated cytosol in the microsome fraction. Addition of cytosol, sulfate ion, and ATP diminished the binding. Parallel experiments using N-hydroxy-2-acetaminofluorene [9-¹⁴C] denied major contribution of this metabolite to the binding of 2-acetaminofluorene in the microsome system. Ring-hydroxylated product was suggested as a possible metabolite for the binding.

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Acetone-fixed smears of DBA/2 mouse leukemia cells that produce clusters of intracytoplasmic A-particles (pronucleocapsids of mouse mammary tumor virus) were employed as an indirect immunofluorescence system to detect the antibody to A-particles in human sera. With positive test sera, specific fluorescence was easily detectable as discrete cytoplasmic granules at the site of A-particle clusters. The antibody was found in 26 (60%) out of 43 breast cancer patient sera and 4 (25%) of 16 mammary fibroadenoma patient sera, while only 4 (11%) out of 37 control woman sera were antibody-positive. In the case of breast cancer patients, occurrence of the antibody was not specifically related to a particular type of tumor histology. In a considerable number of positive cases, the antibody tended to disappear within various lengths of time after surgical operation of the breast cancer.

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A water-soluble nitrosourea, 3-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU, NSC-245382), was tested for its antitumor activity against some kinds of transplantable mouse tumors. The compound was markedly active against myeloid leukemia C1498, plasmacytoma X5563, Ehrlich ascites carcinoma, and mammary tumor FM3A43, and moderately active against mammary tumor MM102 and meningeal sarcoma MS147. It appears that ACNU has a broad antitumor spectrum.

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Immune response in WKA rats immunized with methylcholanthrene-induced KMT-17 tumor was measured by radioisotopic footpad assay. The assay was specific, quantitative, and objective compared with the ordinary method of measuring the thickness of footpads. The strongest footpad reaction was observed 24hr after injection of the antigens. The reaction was transferred by lymphoid cells but not by serum. These results indicate that the footpad assay manifests delayed-type hypersensitivity to tumor antigens.
In order to obtain more reliable antigen preparation for the footpad assay, antigens prepared by several methods were compared. Solubilized antigen extracted with sodium deoxycholate (DOC) showed the strongest reaction in the immunized host and weak reaction in the control non-immunized host. This marked difference between the immunized and control groups indicates that DOC-extracted antigen was better antigen than tumor cells treated with mitomycin-C (40μg/ml), formaldehyde solution (0.2%, 0.01%), X-irradiation (3, 500, 10, 000 rad), non-treated whole-cell antigen, crude membrane, cell homogenates, or antigens extracted by hypotonic sonication and 3M KCl method. The DOC-extracted antigen was well preserved at -20° for 1 month.

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The biochemical basis for natural resistance to 1-β-D-arabinofuranosylcytosine (ara-C) was investigated in the intact cells of 4 rat ascites hepatomas, AH-66F, AH-60C, AH-109A, and AH-66, whose sensitivity to ara-C was different in that decreasing order. The initial rapid uptake of ara-C, mediated by the facilitated diffusion, was similar in all the cell lines tested but the subsequent slow uptake due to phosphorylation of ara-C was inversely correlated with their drug resistance. The capacity for drug phosphorylation was slightly higher in AH-66F and much lower in AH-60C, AH-109A, and AH-66 than in the host bone marrow. In contrast, mouse leukemia L-1210, one of the tumors sensitive to ara-C, phosphorylated the drug about 7 times faster than AH-66F and 4 times faster than the host bone marrow. More than 95% of phosphorylated ara-C was the triphosphate, the active form. Deamination of ara-C was not observed in any tumor or bone marrow. It is concluded that the low capacity for nucleotide formation is related to the natural resistance of rat ascites hepatomas to ara-C.

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The enzyme that phosphorylates 1-β-D-arabinofuranosylcytosine (ara-C) was examined in rat ascites hepatomas to clarify whether the capacity for the phosphorylation of ara-C would be related to natural resistance of the tumors to ara-C. Ara-C kinase activity in AH-66F cells, which were moderately sensitive to ara-C, was approximately the same as that in the bone marrow of tumorbearing rats. L-1210 leukemia, which is sensitive to ara-C, had a higher activity of the enzyme than the mouse bone marrow. On the other hand, the naturally resistant hepatomas, AH-60C, AH-109A, and AH-66, were low in their ara-C kinase activity. The enzyme activity in the cytoplasmic extract (30, 000g supernatant) of the tumor and bone marrow cells was nearly proportional to the capacity for ara-C phosphorylation in intact cells. Thus, the mechanism of natural resistance to ara-C in rat ascites hepatomas was attributed mainly to the low levels of ara-C kinase activity itself. Apparent K_m of the enzyme for ara-C was about 170μM. However, probably due to the presence of the uncompetitive type of inhibitor(s), the level of apparent K_m lowered close to 40μM at higher concentration of the protein as the source of the enzyme, showing similarity to the value in intact cells.

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Tuftsin, a physiological tetrapeptide which stimulates the phagocytic activity of polymorphonuclear neutrophils and macrophages, is found to enhance the migration of normal and sensitized human mononuclear cells. Furthermore, it abrogates the migration inhibition effect of human malignant melanoma antigen. This finding may represent a possible explanation for the unexpected migration enhancement often observed when the migration inhibition test is employed.

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The carcinogenic activity of orally administered N-bis(2-hydroxypropyl)-nitrosamine (DHPN) in male Wistar rats was evaluated with respect to its dose. DHPN was administered at two doses, 100ppm and 500ppm, in the drinking water to rats for 25 to 52 weeks. Tumors developed in the lung, liver, and thyroid of rats receiving 100ppm DHPN and in the lung, liver thyroid, esophagus, kidney, and urinary bladder of rats receiving 500ppm DHPN. The principal target organ was the lung in rats receiving either 100 or 500ppm DHPN, indicating that the carcinogenic action of these doses of DHPN was similar to that of higher doses previously reported. Histologically, the tumors were adenoma, adenocarcinoma, squamous cell carcinoma, and combined carcinoma of the lung, hepatocellular carcinoma and hemangioma of the liver, adenoma and adenocarcinoma of the thyroid, squamous cell papilloma and carcinoma of the esophagus, renal cell and transitional cell carcinoma of the kidney, and transitional cell carcinoma of the urinary bladder. No pancreatic tumors were observed.

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A single intramedullary administration of each dose (15∼20mg) of 4-nitroquinoline 1-oxide, 3-methylcholanthrene, or 7, 12-dimethylbenz[a]anthracene was applied to the mandible, diaphysis, or distal metaphysis of the femur of rabbits. The highest incidence in production of osteosarcoma was obtained from the group in which 4-nitroquinoline 1-oxide was applied to the distal metaphysis (75%, including one case of chondrosarcoma). Tumors hardly appeared in any of the groups when given 3-methylcholanthrene or 7, 12-dimethylbenz[a]anthracene. Histologically, three kinds of entities were recognized from the quantitative difference of the reactive tissues which appeared around carcinogens. It is estimated that the condition of entity III induces the highest incidence of osteosarcoma if chemical carcinogens are given into the bone marrow of experimental animals.

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Distribution of 3-hydroxyanthranilic acid, a tryptophan metabolite suspected of being carcinogenic, was studied in mice. After sc injection of 3-hydroxyanthranilic acid generally labeled with tritium into male (BALB/c × DBA/2)F₁ mice, the distribution of tritium was investigated by whole-body autoradiography and radioactivity measurement. Then, metabolites in the organs and urine were analyzed by paper and thin-layer chromatography.
At 30 and 60min after injection, tritium was distributed mainly in the liver and kidneys, and after 6hr, tritium remained in the lymphoid organs. However, at 30min and 6hr after injection, 3-hydroxyanthranilic acid and its metabolites that might be related to carcinogenic action could not be detected in the liver, kidneys, spleen, or lymph node. 3-Hydroxyanthranilic acid and its sulfate were detected only in urine. These results suggest that 3-hydroxyanthranilic acid is rapidly metabolized and does not accumulate per se in the specific organ. However, urinary organs can be exposed to this compound.

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Association of herpesvirus type-2 (HSV-2) with cervical cancer was studied from seroepidemiological viewpoint, using early antigen induced in Hep-2 cells by HSV-2 4hr after infection (AG-4). The AG-4 antibody was assayed by the microquantitative complement fixation method.
Antibody to AG-4 was respectively detected in 47% and 14% of cervical cancer patients and controls (P<0.001). There was a tendency of the antibody prevalence to increase with clinical stage. There was no correlation between prevalence of AG-4 antibody and that of HSV-2 neutralizing antibody estimated by the kinetics of neutralization assay, confirming previous conclusions that AG-4 differs from viral antigens involved in neutralization. Although previous study could not confirm higher prevalence of neutralizing antibody to HSV-2 in cancer patients, the present data suggest that HSV-2 is not unrelated to cervical cancer.

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Studies were made on the effect of protease inhibitors (pepstatin, leupeptin, and antipain) on the induction of tumors in mice by the α-isomer of 1, 2, 3, 4, 5, 6-hexachlorocyclohexane (α-BHC) and by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Animals were given 0.05% α-BHC in the diet or 0.05% BBN in their drinking water and then fed on a powder diet supplemented with 0.1% protease inhibitors. The incidence of liver nodular hyperplasias was significantly higher in the group treated with α-BHC and leupeptin than in the group treated with α-BHC alone, but pepstatin and antipain did not affect induction of liver nodular hyperplasias by α-BHC. None of the three protease inhibitors influenced the induction of bladder tumors by BBN.

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A powerful immunity to plasmacytoma was produced in mice presensitized with Mycobacterium by the intraperitoneal injection of tuberculin-coated plasmacytoma. Method described here has a wide applicability, because direct coupling tuberculin to tumor cells is very easy.

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Feeding of ochratoxin-A, a mycotoxin, 40ppm in the diet, induced multiple hepatocellular tumors and renal tumors in ddY mice. Pretreatment with aflatoxin-B₁, a single dose of 20mg/kg, enhanced hepatocarcinogenesis of ochratoxin-A but did not affect renal carcinogenesis.

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