Cell kinetic study was performed using labeled mitosis method to compare growth characteristics of androgen-dependent mouse mammary tumor (SC115), which grows only in males, and its subline (Chiba subline No. 1), which has partially lost its dependency during passages through males and grows also in females, although at a slower rate. The subline, compared to the original tumor, showed higher growth fraction irrespective of whether inoculated into a male or a female.
The cell cycle of the subline was faster in males than in females due to acceleration of G1 phase and this trend was reflected in the cell production rate and overall rate of growth. The slower growth in females was restored by the administration of testosterone. However, growth fraction was rather in the inverse relationship with the cell production rate and this fact suggested that androgendeprived conditions do not support survival of non-cycling cells. Primary effect of androgenic environment on this subline was considered to be the reduced length of G1 phase.

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The process and mechanism of peritoneal metastasis of tumor cells were studied experimentally by means of scanning or transmission electron microscopy, employing rat ascites hepatoma AH100B.
Adhesion of tumor cells by microvilli and/or pseudopodia to the mesothelium was observed within 1∼3 days after inoculation when there was no morphological changes of the mesothelial cells. Some changes of the mesothelial cells, such as irregularity, atrophy, and exfoliation, followed tumor cell adhesion 5 or 6 days after inoculation. It was noted that tumor cells adhered to the mesothelium first where no morphological changes were induced, and it is suggested that tumor cells infiltrate into the submesothelial tissue through mesothelial defects.

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The human yolk sac is said to synthesize not only α-fetoprotein but also other serum proteins, i. e., albumin, prealbumin, α₁-antitrypsin, and transferrin. Two endodermal sinus tumors (yolk sac tumors) were successfully transplanted into athymic nude mice. Concentration of these human serum proteins in the sera of the nude mice and tumor extracts was determined quantitatively by electro-immunodiffusion and radioimmunoassay. α-Fetoprotein and albumin were detected in one case, and α₁-antitrypsin and α-fetoprotein in the other. In the former case, the amount of α-fetoprotein synthesized was larger than that of albumin, whereas the amount of α-fetoprotein was smaller than that of α₁-antitrypsin in the latter. It is tempting to consider that human endodermal sinus tumors simulate the human yolk sac functionally, but the pattern of protein synthesis may differ from case to case.

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The effect of a microbial protease inhibitor, leupeptin, on the content of polyamine in the mouse skin was examined during the early stage of tumorigenesis induced by a single application of 7, 12-dimethylbenz [a] anthracene (DMBA) and repeated application of croton oil thereafter. Polyamine content in the skin was measured at 3, 5, 7, and 9 weeks during tumorigenesis. The mice with no visible tumor were selected for measurement of polyamine content at 9 weeks. Mice were left untreated for at least 1 week before measurement of polyamine. Polyamine in the skin was extracted with ice-cold 0.4N HClO₄ and separated into putrescine, spermidine, and spermine fractions through CM-cellulose column. Polyamine concentration was determined by fluorometry with fluorescamine. Group A mice painted with croton oil 3 times a week did not develop tumors. Group B mice painted with a single DMBA developed skin tumors, and group C mice painted with a single DMBA and croton oil 3 times a week showed higher development of skin tumors than group B. Group D mice treated as group C and then painted with leupeptin about 2hr after croton oil treatment developed low average number of tumor. Leupeptin inhibited tumor development. Group A animals sustained high spermidine content at any time tested. Animals in groups B, C, and D had high spermidine content as group A at 3 and 5 weeks. Content of spermidine in group B decreased at 7 and 9 weeks compared with group C which had a high content throughout the time tested. Leupeptin treatment in group D inhibited spermidine content in the skin after 7 weeks without affecting until 5 weeks.

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The effect of BCG, an immunopotentiator, on the hepatocarcinogenesis of the rat induced by 3'-methyl-4-(dimethylamino) azobenzene (3'-Me-DAB) was investigated. After administration of 3'-Me-DAB for 6 weeks, BCG was injected to the rats at 2-week intervals for 50 weeks and the serial determinations of serum α-fetoprotein and morphological examination of the liver were made. In the control group, the second rise of serum α-fetoprotein, which reflected the occurrence of hepatoma, was seen at about 12 weeks after discontinuance of 3'-Me-DAB. On the other hand, the second rise of serum α-fetoprotein in the group treated with BCG was delayed about 10 weeks compared to control group, although there was no significant difference in the final incidence of hepatomas between these two groups. Infiltration of the lymphocytes was observed in the liver of rats treated with BCG without hepatoma. BCG seems to have an inhibitory effect on 3'-Me-DAB carcinogenesis of the rat, possibly by stimulating the cell-mediated immunity, although it is not strong enough to prevent the occurrence of hepatoma completely.

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The metabolism, antitumor activity, and acute toxicity of 5-fluoro-1, 3-bis-(tetrahydro-2-furanyl)-2, 4-pyrimidinedione (FD-1) were investigated in animals, compared with 5-fluoro-1-(tetrahydro-2-furanyl)-2, 4-pyrimidinedione (FT). It was found that after oral administration of FD-1, the level of 5-fluorouracil (5-FU) was maintained higher and longer than after administration of FT, and that a large amount of 5-FU was released from FD-1 by liver microsomal drugmetabolizing enzymes or spontaneous hydrolysis via 5-fluoro-3-(tetrahydro-2-furanyl)-2, 4-pyrimidinedione (3-FT) and FT. FD-1 had a significant activity against the solid form of Ehrlich carcinoma, sarcoma-180, hepatoma AH130, Yoshida sarcoma, Walker carcinosarcoma-256, and leukemia L1210 and P388, but not the ascitic forms, and it produced greater inhibition of tumor growth than FT. The acute toxicity of FD-1 was less than that of FT.

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Combined effect of twenty-one central nervous system depressants with several antitumor agents was studied in the in vitro and in vivo experimental systems, using rat ascites hepatoma call lines, AH13 and AH44, sensitive and insensitive to alkylating agents, respectively. Reserpine remarkably enhanced the cytotoxic effect of 1-(γ-chlorapropyl)-2-chlorornethylpiperidine hydrobromide (CAP-2) both on AH13 and AH44 cells. In the in vivo combined experiments, reserpine also synergistically enhanced the life-prolonging effect of CAP-2 on AH13-bearing rats and, although CAP-2 was not potent on the prolongation of life span of AH44-bearing rats and reserpine was also ineffective at the doses examined, the life span of tumor-bearing rats receiving the combined administration was apparently prolonged compared with control groups. Thus, there was a parallelism between in vitro and in vivo experiments. These findings suggested that the antitumor-enhancing effect of reserpine might be due to the direct action on the tumor cells, and a possible mechanism that reserpine inhibited the DNA damagerepairing activity of the cells was contradictory. Other mechanisms are also discussed.

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An antitumor protein that increased in the serum of OK-432-treated mice showed an enhancing effect on the tumor cell killing by OK-432-elicited macrophages as measured by the ⁵¹Cr release test. This protein, designated as LB, also enhanced the cytolytic effect of macrophages on normal target cells, although the activity was lower toward normal cells than tumor cells. On the other hand, LB exhibited no effect on normal, thioglycolate-elicited, or heat-inactivated OK-432-elicited macrophages.
The effect of LB on the binding of tumor cells to macrophage monolayers was also examined. On the addition of LB, OK-432-elicited macrophages became capable of binding more tumor cells than before. As in the cytolytic assay, the increased binding by the macrophages was not selective for tumor cells, and normal macrophages did not increase the binding of tumor cells upon the addition of LB. These results indicate that LB enhances macrophage-mediated cytotoxicity by increasing intimate binding between the OK-432-elicited macrophages and their target cells.

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Antitumor activities of a combination chemotherapy with a water-soluble nitrosourea, 3-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU), and a single dose of 6-thioguanine were studied using three obstinate murine tumor systems, i. e., Lewis lung carcinoma, B16 melanoma, and an advanced stage of L1210 leukemia systems. Therapeutically synergistic effect was observed either definitely against 1- or 2-day-old Lewis lung carcinoma and 6-day-old L1210 leukemia or moderately against 1-day-old B16 melanoma. Single intravenous treatment on day 7 after subcutaneous implantation of Lewis lung carcinoma, when the tumors had already metastasized to the lungs, produced a significant regression of tumor and a significant increment in survival time of tumor-bearing mice. In comparative studies, the combination of ACNU and 6-thioguanine showed a greater and a wider spectrum of antitumor activities against these tumors than those obtained by the combination with ACNU and a single dose of 5-fluorouracil, methotrexate, or 6-mercaptopurine. Increment in lethal toxicity for normal and tumor-bearing mice was not observed by the combination of ACNU and 6-thioguanine in contrast to definite increases in this toxicity by the combination of ACNU and 5-fluorouracil. The present experimental results may suggest the clinical utility of the combination chemotherapy with ACNU and 6-thioguanine in the treatment of several solid tumors as well as acute leukemias.

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Human adherent cells from peripheral blood were cultured with immunostimulant, BCG, yeast cell wall, or streptococcal preparation (OK-432), for 3 days, and the cytostatic activity of the adherent cells on human tumor cells was examined. The cells cultured in the presence of an immunostimulant exhibited increased phagocytic activity and the number of phagocytosed sheep red blood cells (sRBC) per cell increased. Adherent cells cultured without the immunostimulant showed slight cytostatic activity of 8∼20%. HD-10 cells, derived from Hodgkin's disease, and QG-K and QG-U cells derived from uterine cervical cancer were more susceptible than HeLa cells to the adherent cells activated by OK-432 or yeast cell wall. Relationship between population doubling time and susceptibility to the cytostatic effect mediated by the activated adherent cells was not observed. The supernatant from the activated adherent cells was also effective in inhibiting DNA synthesis of the rapidly proliferating target cells, HD-10 cells and HeLa cells. However, the proliferation of the other two cell lines was enhanced. The effect of activated adherent cells on tumor cell proliferation and its relation to cytostasis were examined and discussed.

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Friend leukemia cells were adapted to grow in a medium containing 0.5% serum by gradually decreasing concentration of the serum from 15%. Upon treatment of the cells with dimethyl sulfoxide, appearance of benzidine-positive cells was suppressed in the cells cultivated in a 0.5% serum medium, whereas globin mRNA sequences, as determined by hybridization of cytoplasmic RNA with globin cDNA, increased to the same order as in the cells grown in a medium containing 15% serum. A small but significant amount of globin was synthesized in cells treated with dimethyl sulfoxide in 0.5% serum medium, as determined by dodecyl sulfate-polyacrylamide gel electrophoresis of ³H-labeled cellular proteins.

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An in vivo stathmokinetic technique with vincristine sulfate (VCR) was used to analyze the mitotic activity of carcinoma cells from 53 gastric cancer patients, and the correlation between mitotic activity and various states of cancer was studied.
In the metaplastic mucosa, almost all mitotic cells (96.4%) were found in the lower half segment of the mucosa. Therefore, the segment was thought to be the proliferative area. In the cancerous lesion as well as in the metaplastic mucosa, VCR produced increase in mitotic index permill (MI) due to the increase in mitotic cells at metaphase. After a single dose (0.02mg/kg body weight) of VCR, a rapid and linear increase in MI was found in the metaplastic mucosa during 16hr. The average value of MI analyzed at 16hr after VCR administration was 97.3‰ in the metaplastic mucosa and 84.1‰ in the cancerous lesion. Average cell production rate (CPR) was estimated as 4.6 cells/1, 000cells/hr in the metaplastic mucosa and 3.9cells/1, 000 cancer cells/hr in the cancerous lesion.
Cancerous lesions showed changes of MI in various states; the increase in MI paralleled increase in the diameter, degree of stages, and in invasive layers of the gastric wall. The average MI of early cancer was 60.1‰ and that of advanced cancer was 95.0‰. Poorly differentiated adenocarcinoma revealed a higher value of MI (average, 91.0‰) than that (average, 78.1‰) of differentiated adenocarcinoma. Metastatic lesions also showed a higher value of MI (average, 120.1‰) than that (average, 95.0‰) of primary lesions. However, there was no correlation between the average values of MI and the location, degree of the stromal reaction, or types of infiltrative growth in the cancerous tissue, and age and sex of the patients.

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N-Amyl-N-methylnitrosamine (AMN) induced esophageal carcinoma in Donryu strain rats when given in drinking water. The incidence of carcinomas was high when AMN was given as 0.0015% solution for 90 days or 0.003% solution for 60 days. There was no difference in the incidence in males and females. Sequential studies showed that 4-week administration was necessary to induce esophageal papillomas, and 8-week administration to induce esophageal carcinomas. Whole-body autoradiography after intravenous injection of N-amyl-N-methyl [¹⁴C]-nitrosamine (¹⁴C-AMN) indicated that AMN accumulated preferentially in the esophagus.

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The antitumor activity of 1-(2-tetrahydrofuryl)-5-fluorouracil (FT-207) on sarcoma was enhanced by oral coadministration of uracil, thymine, or thymidine. The activity was enhanced equally by thymine and by uracil than by thymidine, but thymine caused loss in body weight. The antitumor activity of 5-fluorouracil (5-FU) was also enhanced by thymine or uracil, but both caused loss in body weight. Degradation of 5-FU in vitro was inhibited more by thymine than by uracil. Phosphorylation of 5-FU, however, was not inhibited by uracil, thymine, or thymidine, even at 100 times the concentration of 5-FU. These results suggest that the mechanism of enhancement of the antitumor activity of FT-207 by thymine or thymidine was similar to that by uracil, and that uracil had more effect than thymine or thymidine in enhancing antitumor effect of these drugs to FT-207 without toxicity.

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Antitumor effect against mouse Ehrlich ascites carcinoma was examined on Neocarzinostatin (NCS) entrapped in sonicated liposomes (neutral and negatively or positively charged forms) in comparison with free NCS. In the intraperitoneal inoculation-intraperitoneal medication system, NCS entrapped in negatively charged liposomes, which was most unstable in aqueous solution, showed statistically significant prolongation of survival days in mice compared to free NCS. In contrast, survival days of the animals given NCS entrapped in positively charged liposomes were shorter than those of the corresponding groups given free NCS. On the other hand in the intraperitoneal inoculation-intravenous medication system, survival days were not prolonged markedly by administration of free NCS, but when NCS was entrapped in positively charged or neutral liposomes, prolongation of survival was apparent compared to the corresponding groups given free NCS. The most marked effect was seen when NCS was entrapped in positively charged liposomes, which show the best stability in aqueous solution.

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5'-Deoxy-5-fluorouridine (5'-DFUR) was parenterally and orally offective on various transplantable tumors and its activity was better than that of other fluorinated pyrimidines. However, like 5-fluorouracil and 2'-deoxy-5-fluorouridine (FUdR), 5'-DFUR was ineffective on L1210 leukemia resistant to 5-fluorouracil, suggesting that it would exert its antitumor activity through converted 5-fluorouracil. In tissue culture, 5'-DFUR inhibited the growth of various tumor cells similarly to other fluorinated pyrimidines. However, 5'-DFUR was unique in that uridine completely reversed its inhibitory effect. Enzymological study clarified that uridine inhibited the conversion of 5'-DFUR to 5-fluorouracil by a uridine phosphorylase, in parallel to its reverse effect on cell growth inhibition by 5'-DFUR. Furthermore, a subline of L1210 leukemia resistant to 5'-DFUR but not to 5-fluorouracil was found to lack the uridine phosphorylase. These results indicate that 5'-DFUR is a depot form of 5-fluorouracil which can be promptly activated by uridine phosphorylase. In addition, the uridine phosphorylase was found to be abundant in sarcoma-180 solid tumor, leading to a significantly higher concentration of converted 5-fluorouracil in this tumor than in other normal tissues. This provides a good explanation for the high chemotherapeutic index of 5'-DFUR against this tumor, which may be applicable also for other tumors.

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Mutagenic and DNA-damaging effects of a series of N, N-dialkylnitrosamines having an α-acetoxy group together with an α-acetoxy or an ω-methoxycarbonyl group were tested in Salmonella typhimurium, Escherichia coli, and Bacillus subtilis without metabolic activation. The compounds comprised each of 3 N-(ω-acetoxyalkyl)-N-(acetoxymethyl) nitrosamines, N-(ω-methoxycarbonylalkyl)-N-(acetoxymethyl) nitrosamines, and N-(ω-methoxycarbonylalkyl)-N-(α-acetoxybutyl) nitrosamines. All the compounds gave positive results in these mutagenicity and repair tests. No definite differences were observed in the mutagenic and DNA-damaging effects between the model compounds derived from the carcinogenic compounds and those derived from the non-carcinogenic counterparts. The strongest mutagenic activity was observed so far with N-(2-methoxy-carbonylethyl)-N-(1-acetoxybutyl) nitrosamine in all of the four bacterial strains.

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Two out of 9 weanling hamsters, treated with intravenous inoculation of highly concentrated human papovavirus BK, propagated in human embryonic kidney cells, developed osteogenic sarcomas of the costal cage and the mandibula. The sera of the tumor-bearing animals were positive for BK T-antigen and the successively transplanted tumors were positive for intranuclear T-antigen when tested with anti-SV40 T-antibody by immunofluorescence. The tumor tissue exhibited various stages of maturation, from areas showing immature sarcoma and angiomatous sarcoma to those of well-differentiated osteogenic sarcoma. Tumor histology was described in detail.

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Male Wistar rats were given a diet containing 0.1% leupeptin, a microbial protease inhibitor, and drinking water containing 0.01% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) for 8 weeks. Leupeptin did not have any detectable effect on the induction of bladder tumors by BBN in week 40, in terms of the weight of the bladder including the tumor, the number of tumors per bladder, the extent of invasion, or the incidences of hyperplasia, papilloma, and cancer.
This result is in marked contrast to the previous findings that leupeptin enhanced BBN-induced bladder carcinogenesis when administered continuously after BBN-treatment.

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The effect of chloroquine on differentiation of cultured mouse myeloid leukemia Ml cells was examined. On treatment with 5∼25μg/ml of chloroquine diphosphate for 1∼4 days, the cells were induced to phagocytize latex beads, to form Fc rosettes, to form dispersed colonies in soft agar, and to synthesize lysozyme, unlike untreated cells. The morphology of about 40% of the cells changed during treatment with 20μg/ml of chloroquine diphosphate for 4 days; some cells developed small eccentrically located nuclei, and others ring-shaped or segmented nuclei. These results show that Ml cells differentiate into cells resembling macrophages or granulocytes on treatment with chloroquine.

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The isozyme patterns of glycolytic enzymes of Friend leukemia cells (FLC) were compared with those of erythrocytes and erythroblasts. Erythrocyte-specific R types of pyruvate kinase (PK) were clearly observed in phenylhydrazineinduced mouse erythroblast, and much less amount of them was also observed in Friend leukemia cells. When FLC were induced to differentiate by hexamethylene-bisacetamide (HMBA), the R types were slightly reduced. When the induction of differentiation was inhibited by 12-O-tetradecanoylphorbol 13-acetate (TPA), the R types and M₂-R hybrids rather increased. These results are reverse of those obtained when hemoglobin production is used as a marker of differentiation. Isozyme patterns of lactic dehydrogenase and aldolase did not change during differentiation of FLC induced by HMBA, and were the same as those of mouse erythroblasts and erythrocytes.

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The growth of subcutaneously transplanted Lewis lung carcinoma in BDF₁ mice was inhibited by an inorganic dye, Ruthenium Red. The mean volume of the tumors of the mice which were given Ruthenium Red by daily ip injection of 5mg/kg on days 1∼10 was 28 and 39% of that of the controls at days 18 and 23 after transplantation, respectively. The median survival time (MST) of the treated mice was 27.8 days while that of the control was 23.0 days. The higher MST (32.5 days) was obtained when the dye was given at 2.5mg/kg for 1∼20 days, although the tumor volume in this group of mice was 51% of that of the control at day 18.

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