Treatment with cycloheximide and a hepatotoxic dose of CCl₄ (well tolerated in normal animals) is acutely toxic in animals bearing the Ehrlich ascites tumour. Reduction in tumour load 4hr after the administration of cycloheximide and CCl₄ is life-saving: treatment with a hepatotoxic dose of CCl₄ 30hr before challenge with cycloheximide and CCl₄ also nullifies that otherwise life-threatening treatment.

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To investigate the influence of operative stress on tumor growth, laparotomy and/or thoracotomy were performed in association with intraperitoneal and intravenous inoculation of Sato lung cancer cells into Donryu rats. Survival time and the number of metastatic nodules on both lungs were examined. Five-hour laparotomy and/or one-hour thoracotomy were performed on day 2 after inoculation. The enhancing effects on tumor growth of a five-hour laparotomy and of a one-hour thoracotomy were similar as regards survival time, the rate of 50-day survivors and the number of metastatic nodules on the lungs. Thoracotomy seems to enhance tumor growth several times more effectively than laparotomy. The addition of a bleeding procedure (4ml) to these operations had little effect on the tumor growth. As for the operation timing, it was found that the operative stress of thoracotomy or laparo-thoracotomy enhanced tumor growth most markedly when the operation was performed on day -1. The enhancing effects were almost the same, and the difference from the control was still highly significant, when the operation was performed on days -2, 0 or +2.

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The present series of experiments was carried out in order to see what role pre-existing localized fibrosis plays in carcinogenesis of the lung.
Hemorrhagic infarction was produced in the lung of 180 male Wistar rats by injecting 0.05ml of hexachlorotetrafluorobutane into the tail vein. This resulted in localized fibrosis in the lung 3 months later. One hundred and fifteen rats were alive 3 months after administration of the chemical. Of these animals, 30 were given no further treatment (control). The remaining 85 rats were given intracheal instillation of 0.2μCi of polonlum-210 once a week, a total of 15 times.
It was subsequently found that lung carcinoma was induced in close proximity to the localized pulmonary fibrosis in 3 of 26 rats (11.5%) during the period from completion of the 15 weekly administrations of polonium-210 until the end of this experiment (21 months after the 1st instillation of polonium-210). Polonium-210 was found to be deposited in the fibrous thickening of the alveolus around the subpleural fibrotic lesion, bronchial epithelium, and peribronchial lymph apparati at the initial period of administration of polonium-210, but during the period of pulmonary carcinogenesis, it was deposited in the localized fibrotic lesion in the lung and in a few cancer cells.
This suggests that polonium-210 deposited in the pulmonary fibrotic lesion remains there over a long period of time, indicating a reduced clearance ability at this site.

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The effect of thioglycollate-induced macrophages on the growth of syngeneic B16 melanoma cells was studied. Thioglycollate-induced macrophages showed strong cytostasis and cytotoxicity towards B16 tumor cells in vitro. However, these effects were not demonstrable in an in vivo assay which involved local transfer and experimental metastasis. In contrast to the in vitro results, mice injected iv with both B16 tumor cells and thioglycollate-induced peritoneal exudate cells developed a significantly higher number of lung metastases than mice injected with B16 tumor cells alone. Possible reasons for the failure of thioglycollate-induced macrophages to exhibit cytostatic and cytotoxic activity in vivo are discussed.

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The characteristics of WI-38 CT-1 cells, which had been transformed by treatment with Co-60 gamma rays, were compared with those of the control WI-38 cells in order to identify parameters of malignant transformation in cultures of normal human diploid cells. The transformed WI-38 CT-1 cells were different from the control WI-38 cells in the following respects: (1) epithelial-like morphology of the cells, (2) reduced serum requirement for cell proliferation, (3) increased colony formation in soft agar, (4) increased saturation density, and (5) resistance to the cytostatic effect of theophylline on cell proliferation. On the other hand, no appreciable difference was detected between the control and the transformed cells in (1) population doubling time and (2) sensitivity to Co-60 gamma rays.

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A new anthramycin-group antitumor antibiotic, neothramycin, isolated from a Streptomyces culture, showed activity against experimental tumors such as lymphocytic leukemia P388, ascites sarcoma-180, hepatoma AH130, Walker carcinosarcoma-256 and mouse mammary adenocarcinoma (CCMT). In particular, the tumor growth inhibition ratio was as high as 96% when neothramycin was injected intraperitoneally at a daily dose of 2mg/kg into Wistar strain rats previously inoculated with Walker carcinosarcoma-256 by the subcutaneous route. Successive intraperitoneal injections of the compound were more effective than a single injection with the P388 system.

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A human adenocarcinoma cell line was successfully established from the lymph-node metastasis of transverse colon cancer. Primary culture was done with the tumor developed in a nude mouse which had been inoculated with the metastatic lymph node. Morphological characteristics, growth kinetics, chromosomes, tumorigenicity and high production of carcinoembryonic antigen by the established cells are described. This cell line should be a useful tool for studies on the mechanism of re-expression of the fetal gene which is responsible for carcinoembryonic antigen expression in colon cancer cells.

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A simple method to establish hypoxic conditions in vitro is described and the cytotoxicity and radiation dose-modifying effect of misonidazole were investigated, using HeLa S3 cells. Under hypoxic conditions, 5-hr exposure to 50mM misonidazole resulted in severe cytotoxicity, though the toxicity at 1mM was low. The survival curves of cells exposed to 5 or 10mM misonidazole showed an initial exponential portion followed by a less steep decline. Under aerobic conditions, exposure to 1mM for 5 days showed no cytotoxicity, while the surviving fraction of cells exposed to 50mM for 10hr was reduced to 0.3. Acute conversion from hypoxic to aerobic conditions revealed persistence of the cytotoxicity for 2hr after the conversion. Under hypoxic conditions, a dose-modifying factor of 1.9 was achieved for cells irradiated with gamma rays.

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Using macrophage migration inhibition (MMI) and the cell-mediated cytotoxicity (CMC) test, the cell-mediated immune response in hamsters with transplanted HVJ-carrying tumor cells (THEL-HVJ or THEL-HVJ_pits) was examined in relation to the lowered transplantability of the cells. The cells cultured at a temperature (34°) permissive for HVJ_pits (temperature-sensitive HVJ) showed significantly lowered transplantability in hamsters. After shifting the cell culture temperature up to 39° (non-permissive for HVJ_pits) for 5 days, THEL-HVJ_pits cells which had lost their cellular HVJ antigens regained the same high transplantability observed in parent THEL cells. However, culture of the cells at 37° (partially permissive) for 24hr did not significantly affect their tumor-forming ability with lowered transplantability, in spite of a considerable reduction in cellular HVJ antigens.
The MMI test on hamsters with transplanted THEL-HVJ or THEL-HVJ_pitscells cultured at 34° (MMI/THEL-HVJ 34 or MMI/THEL-HVJ_pits 34) was more markedly positive, and for a longer period (1 to 4 weeks), than the same test on hamsters inoculated with THEL cells. However, the MMI/THEL-HVJ_pits 39 cells acquired lowered reactivity like those from THEL-tumor bearing animals, while MMI/THEL-HVJ_pits 37 cells were still positive, just as in MMI/THEL-HVJ_pits 34. In the CMC test, much more cytotoxic activity was observed in spleen cells from hamsters with transplanted THEL-HVJ_pits cells than in those from THEL-transplanted animals; there was a general correspondence with the results obtained in the above MMI test.
These findings strongly indicate that the lowered transplantability of HVJ-carrying tumor cells may be due to a significant induction of cell-mediated immune responses. It is suggested that cell membrane antigens modified (xenogenized) by the complete or partial expression of HVJ genomes carried may play an important part in this induction in vivo.

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The inhibitory effect of an aromatic retinoic acid analog, ethyl all-trans-9-(4-methoxy-2, 3, 6-trimethylphenyl)-3, 7-dimethyl-2, 4, 6, 8-nonatetraenoate, on bladder carcinogenesis in rats treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was evaluated. Administration of 50 ppm of aromatic retinoid in the diet before BBN in the drinking water reduced the incidence of papillary or nodular hyperplasia as a preneoplastic lesion of the bladder epithelium (P<0.05). When given before, during or after BBN, it also greatly reduced the incidence of papilloma (P<0.001 before BBN, P<0.01 during or after BBN treatment), and slightly inhibited the development of cancer. Administration of 100ppm of aromatic retinoid before or during BBN administration also reduced the incidences of papillary or nodular hyperplasia (P<0.01 before BBN, P<0.05 during BBN treatment), and its administration before, during or after BBN treatment greatly reduced the incidences of papilloma (P<0.001), and cancer (P<0.01 before or after BBN, P<0.001 during BBN treatment). Similar results were obtained by assessing the effect of the retinoid on the average numbers of various epithelial lesions per 10cm length of basement membrane of the bladder in tissue slices. These results show that the aromatic retinoid inhibits both the initiation and promotion of bladder carcinogenesis induced in rats by BBN, and that its effect is dose-dependent.

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The reversible inhibitory action of an aromatic analog of retinoic acid, ethyl all-trans-9-(4-methoxy-2, 3, 6-trimethylphenyl)-3, 7-dimethyl-2, 4, 6, 8-nonatetraenoate (aromatic retinoid) on bladder carcinogenesis was studied by biochemical and histopathological methods. Fischer 344 female rats were fed a diet containing the aromatic retinoid (0, 12.5 or 50ppm) for 56 days. On day 57, 60 or 70, 50mg/kg body weight of N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN) in a volume of 0.3ml was directly instilled intravesically through a urethral catheter. DNA damage in rat bladder epithelial cells 2, 24 and 48hr after administration of BCPN was examined by measuring the change in sedimentation profile of the DNA in 5∼20% alkaline sucrose gradients. DNA sedimentation was measured by a fluorometric procedure. Histopathological examination of the bladder epithelium was performed at the same time. Two hours after a 5-min exposure to BCPN, DNA damage at the urothelium was observed in rats fed the diet without the aromatic retinoid, but the damage was repaired after 24 and 48hr. The highest dose of the aromatic retinoid almost completely prevented DNA damage by BCPN on days 57 and 60. A lower dose of the aromatic retinoid did not prevent DNA damage by BCPN on days 57, 60, and 70, while the higher dose did not prevent damage by BCPN on day 70.
No histological changes were observed in the urothelium of rats treated with the aromatic retinoid and BCPN, and electron microscopy showed that the epithelium also remained normal.
DNA damage was induced by BCPN at the urothelium, and the damage was inhibited by previous administration of the aromatic retinoid.

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Studies were made on the chemotherapy of gastric cancer in dogs induced by N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Four male Beagle dogs were given a solution of ENNG at 100∼150μg/ml with or without 0.4% Tween 60 to drink for 5∼8 months. They all developed gastric adenocarcinomas, which were confirmed by histological examination of biopsy specimens taken in months 13∼32 of the experiment. After confirming the presence of gastric cancer, 1-n-hexylcarbamoyl-5-fluorouracil (HCFU), a derivative of 5-fluorouracil, was given to the dogs orally as capsules at a daily dose of 5 or 10mg/kg body weight. One dog died from adverse effects of HCFU 12 days after the beginning of chemotherapy. The other 3 dogs were treated with HCFU for 82∼424 days. In these dogs, the tumor size, measured by X-ray examination, increased during chemotherapy. On autopsy, the tumors in the stomach were found to be restricted to the antrum, and metastases of the gastric adenocarcinomas to the regional lymph nodes and/or liver were found in 2 dogs. No degenerative changes of tumor cells were found in the stomach or metastasized organs, except for necrosis of cells in a perigastric regional lymph node of one dog. The value of using canine gastric cancer in studies on chemotherapy is discussed.

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Recent studies have demonstrated that nitroso chemical carcinogens markedly activate guanylate cyclase, which catalyzes the production of guanosine 3', 5'-monophosphate (cyclic GMP). We therefore examined the effect of inhibitors of carcinogenic compounds on guanylate cyclase activation by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). An antioxidant group of anticarcinogenic compounds was effective. Disulfiram and phenethyl isothiocyanate exhibited the most potent inhibition. Inhibitor constants (Ki) for disulfiram and phenethyl isothiocyanate were 1.2×10^-5M and 4.9×10^-5M, respectively. Sodium diethyldithiocarbamate, phenyl isothiocyanate, butylated hydroxyanisole and ethoxyquin showed moderate inhibitory effects. Sodium selenide decreased the MNNG-activated guanylate cyclase activity to about 30%, and it was inhibitory at the low concentration of 10^-5M. The present data suggest that one of the mechanisms by which anticarcinogenic compounds exert their effect may in part be related to the inhibition of guanylate cyclase.

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Changes in the level of γ-glutamyl transpeptidase (γ-GTP) in the rat liver with age were studied histochemically and biochemically. A high enzyme activity was seen throughout the hepatic lobule with some predominance in the periportal area in the neonatal rat liver. This enzyme activity disappeared almost completely from the hepatocytes of young adults. However, after 30 weeks of age, there was a gradual re-elevation of the enzyme activity in the periportal hepatocytes, attaining a level similar to that of neonatal liver. Expression of γ-GTP activity in the rat hepatocytes is associated with not only onconeonatal events but also aging.

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Mason-Pfizer monkey virus (MPMV), baboon endogenous virus (BaEV) and simian sarcoma virus associated virus (SSAV) induced fusion in cultured human cells derived from tumors or transformed in vitro. Not only virus-transformed cells, but also cells transformed spontaneously by 4-nitroquinoline 1-oxide (4-NQO) treatment or by ⁶⁰Co-gamma ray irradiation were fused by these simian retroviruses. Non-transformed cells derived from normal human embryos were not fused by any of these viruses. Of these tumor or transformed cells, cells carrying Rous sarcoma virus (RSV) genome were most extensively fused by these simian retroviruses. Anti-MPMV, anti-BaEV and anti-SSAV sera blocked the cell fusion mediated by MPMV, BaEV and SSAV, respectively, and not that by other viruses, indicating that the cell fusion was virus-specific.

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The saturation hybridization levels of DNA with rRNA (hereafter abbreviated as rDNA/DNA) in adult liver, bone marrow, and fetuses in Long-Evans rats were 0.042, 0.036, and 0.029∼0.033%, respectively, showing a lower level in rapidly growing tissues. We consider that the lower rDNA/DNA in rapidly growing tissues might be due to the replication of rDNA in the late S phase, or in other words, to the depression of rDNA/DNA in each S phase. Based on these findings, the relative levels of rDNA per G₁ genome were estimated in cells of these normal tissues and 4 diploid tumors by multiplying rDNA/DNA and DNA/genome. By this procedure, rDNA/genome was shown to be constant in normal G₁ cells, while a 31∼48% increase was found in the tumor cell genome. Direct measurement of rDNA in synchronized #2 trisomy leukemia cells confirmed the replication of rDNA in the late S phase, with a 21.4% increase of rDNA per G₁ genome.

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Using a culture line of renal carcinoma of Syrian hamster (HKC-400), the distribution of DNA content in individual cells was analyzed by autoradiography after pulse and continuous labeling with ³H-thymidine in order to investigate the behavior of the non-proliferative cell compartment.
Additional data used were the cell cycle parameters obtained by the fraction of labeled mitoses (FLM) method and the growth rate. The principle of analysis is to compare the cell fractions having G₁ and G₂ DNA contents with the fractions of G₁ and G₂ phase cells. It was concluded that even in the exponential phase of growth 31% of cells were blocked in either the G₁ or G₂ DNA region (Q₁=22% and Q₂=9%), and the cell loss rate from Q₁ was higher than from Q₂.

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The leucocyte adherence inhibition (LAI) microassay using crude extracts from colorectal cancer cell lines (M-7609 and S-7512) was evaluated as a test of immunological antitumor reactivity in colorectal cancer patients. The optimal antigen concentration for differentiating cancer patients from control subjects was 0.05mg/ml protein. The cut-off value of LAI index for determining reactive and nonreactive cases was -0.20. The numbers of colorectal cancer patients reactive against antigens M (derived from M-7609), S (derived from S-7512) and M+S were 9 of 19, 9 of 19 and 12 of 18, respectively, whereas against control antigens K (derived from K-7610, pulmonary cancer), SF (derived from cultured fibroblastic cells) and T (derived from fresh colorectal cancer tissue), they were only 1 of 18, 1 of 17, and 3 of 15, respectively. The mean LA indices of the Dukes A and B group, and of the Dukes C and recurrent cancer group were -0.28 and -0.24, but that of a tumor-free postoperative group, another malignant disease group and a group of healthy individuals were -0.04, -0.05, and -0.03, respectively. Judging from these results, LAI microassay using cultured human cancer cell lines as tumor antigen sources is suitable as a test for evaluating the antitumor immunoreactivity of colorectal cancer patients.

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The polyamine content in erythrocytes was determined in an attempt to establish a biological marker of tumor growth in patients with malignant lymphoma. Erythrocytes from 24 patients with malignant lymphoma and 40 healthy controls were subjected to polyamine determination, using high-performance liquid chromatography. The spermidine and spermine levels in normal human erythrocytes were 15.06±3.61 and 8.79±3.12nmol/10¹⁰ cells, respectively. Spermidine and spermine levels in erythrocytes of lymphoma patients were 14.16±4.99 and 13.06±5.37nmol/10¹⁰ cells in the stage I and II groups, 21.25±3.80 and 14.17±6.11 in the stage III group, and 34.17±10.37 and 24.39±16.08 in the stage IV group, respectively. The levels of both polyamines were significantly elevated (P<0.005) in erythrocytes of stage IV patients, while those of stage I and II patients were within the normal range.
Serial studies in 8 patients indicated that the levels of both polyamines in erythrocytes correlated well with the clinincal state. Thus, the determination of polyamines in erythrocytes should be clinically useful to determine the state of progression of malignant lymphoma.

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Repeated intravenous administrations of oil-attached Nocardia rubra cell-wall skeleton (N. rubra-CWS) prevented the induction of lung cancer by intrabronchial instillations of 3-methylcholanthrene in rabbits. Amyloidosis was seen after giving 3-methylcholanthrene, and the incidence was higher in the N. rubra-CWS administered rabbits than in the controls.

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Segments of surgically resected human adult bronchi were xenotransplanted into the subcutaneous tissue of 35 BALB/c genetic background athymic nude mice with 3-methylcholanthrene placed in their lumina. Bronchial segments were removed 8 to 20 weeks after transplantation and preservation of the bronchial mucosa was confirmed in 26 segments. The most frequent change observed in the mucosa was goblet cell hyperplasia, followed by squamous metaplasia and dysplasia. Severe dysplasia or questionable squamous cell carcinoma in situ was noted at the site of the bronchial gland in a segment removed 8 weeks after transplantation, and invasive squamous cell carcinoma in a segment removed 15 weeks after transplantation. A difficulty in this work was the frequent earlier development of malignant tumors of mouse origin.

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The antileukemic combined effect of reserpine with 1-γ-chloropropyl-2-chloromethylpiperidine hydrobromide (CAP-2) and other antitumor agents was studied on mouse leukemia L1210 in comparison with the effects of other Rauwolfia alkaloids and sympatholytic drugs. When reserpine was administered by a single ip injection (2.5mg/kg) on day 1, the effect of subsequent administration of CAP-2, mitomycin-C, or vinblastine was synergistically enhanced. In this combination with reserpine, the acute lethality of CAP-2 on the host animals was apparently decreased. Among other sympatholytic drugs, rescinnamine, a central nervous system depressant, slightly potentiated the antitumor effect of CAP-2. On the other hand, when reserpine-induced hypothermia was prevented by maintenance of the ambient temperature at 30°, the synergistic combined effect of reserpine was diminished. Chlorpromazine-induced hypothermia did not influence the antitumor effect of CAP-2. It may be concluded that the antileukemic synergism depends partially on the interaction between reserpine and CAP-2 or other antitumor agents in relation to body temperature and/or action on the central nervous system in leukemic mice.

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