The hepatic cells of rats being fed acetylaminofluorene were transferred into culture at various times. Proliferative hepatocytic foci were obtained from animals treated with the carcinogen for more than 9 weeks. These hepatocytic foci became transplantable and capable of growth in soft agar after 4 to 12 months in culture. Phenobarbital markedly enhanced the growth of these hepatocytes in culture.

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The mechanisms of inhibition by simultaneously administered phenobarbital of 3'-methyl-4-(dimethylamino) azobenzene (3'-Me-DAB)-induced hepatocarcinogenesis in the rat were studied. Weanling rats were fed a diet containing 0.06% 3'-Me-DAB or 0.06% 3'-Me-DAB and 0.05% phenobarbital for 3 weeks, followed by either basal diet or a diet containing 0.05% phenobarbital as a promoter. The number and the size of enzyme-altered islands and the number of tumors larger than 5mm in diameter were scored at week 12 and week 40, respectively. The simultaneous feeding of phenobarbital and 3'-Me-DAB resulted in a significant decrease in the number and size of enzyme-altered islands and in the number of tumors, in comparison with those scored in animals fed 3'-Me-DAB alone. It was concluded that the simultaneous feeding of phenobarbital inhibits both the initiation of carcinogenesis and also the promotive action of the carcinogen resulting from its selective toxicity on the liver tissue.

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As an approach to the development of new antitumor agents, a hybrid in which one molecule of the Fab' fragment of a rabbit anti-murine leukemia L1210 immunoglobulin G (IgG) was linked to the A-chain of ricin via a disulfide bond was prepared by the reaction of A-chain having one reactive thiol group with Fab' having one activated cysteine residue, followed by chromatography on Sephadex G-150 superfine. The hybrid exhibited a potent cytotoxicity towards L1210, whereas unconjugated Fab', unconjugated A-chain, or an equimolar mixture of the two showed no significant cytotoxicity. Furthermore, the hybrid with Fab' of normal IgG had no cytotoxicity towards L1210 cells. These results indicate that the hybrid manifests its toxic activity towards the target cells through binding of its Fab' moiety to cell-surface antigens.

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As a model experiment for the preparation of cancer chemotherapeutic agents, a disulfide-linked conjugate of rabbit anti-mouse IgG (RaMIgG) and ricin A-chain was synthesized. Activated sulfhydryl groups were first introduced into RaMIgG and then the modified RaMIgG was reacted with ricin A-chain. The fraction containing the conjugate was obtained from the reaction mixture by gel filtration through Sephadex G-150 and was characterized by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis and double immunodiffusion in agar. The inhibitory activity of A-chain against protein synthesis in a cell-free system and the binding activity of RaMIgG to mouse B lymphocytes were both fully retained. The purified conjugate selectively inhibited protein synthesis in mouse B lymphocytes possessing cell surface Ig, but not that in thymocytes. The cytotoxicity was inhibited by adding mouse IgG and an excess amount of RaMIgG to the cell culture. The effects of unlinked RaMIgG and A-chain on the cytotoxicity were negligible even when the two were used as a mixture.

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Temperature-sensitive (ts) variants that express a transformed phenotype at low (33°) but not at high (38.5°) temperature were isolated from a mouse fibroblast strain C3H2K cells. Among these variants, cloned ts-12B cells showed at 38.5° a density-dependent inhibition of growth typical of normal fibroblasts cultured in vitro. However, at low temperature they lost this capacity and grew to a higher saturation density. The ts variant was also temperature-sensitive as regards serum requirement: it required a higher concentration of serum for growth at 38.5° than at 33°. However, the cells behaved at both temperatures like the parent strain, possessing anchorage-dependence for growth and fibronectin, whereas they were like transformed cells with respect to release of high fibrinolytic activity. No type-C virus core protein p30 was detected at either temperature. Thus, various parameters of transformation in vitro were independently regulated in these variant cells.

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Peripheral blood lymphocytes from both normal donors and gastric cancer patients contained suppressor cells which could be activated by concanavalin A (Con A) to suppress the proliferative response of lymphocytes from a normal donor. Con A-activated suppressor cells resided in the E rosette-forming cell fraction. An apparent inverse relationship was found between Con A-activated suppressor cell activity and lymphocyte proliferative response to phytohemagglutinin. Significant increases of suppressor cell activities were found in advanced gastric cancer patients. These activities decreased remarkably after surgical resection of the primary tumors. It is likely that nonspecific suppressor cells represent one of the major factors inducing immunosuppression in gastric cancer patients.

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The antitumor activity of saframycin was examined against four different experimental tumor systems in mice. Saframycins A and C inhibited the growth of L1210 cells in suspension culture completely at concentrations of 0.02μg/ml and 1.0μg/ml, respectively. The LD₅₀'s of saframycin A for ddY mice were 4.9mg/kg (ip) and 3.3mg/kg (iv), respectively. In C3H/He mice, the LD₅₀'s were 10.5mg/kg (ip) and 9.7mg/kg (iv), respectively. Saframycin A was highly active against Ehrlich ascites carcinoma and P388 leukemia, and moderately active against L1210 leukemia and B16 melanoma. The antitumor activity of saframycin A was 50 to 100 times greater than that of saframycin C. The survivors cured of Ehrlich ascites carcinoma by treatment with saframycin A developed a resistance to rechallenge with the same tumor. On the other hand, when carbazilquinone and adriamycin were used as reference drugs, the cured mice in these cases did not resist rechallenge with the same tumor. When saframycin A (5mg/kg) was administered intraperitoneally into mice, the blood concentration of saframycin A was 4.6μg/ml after 30min, and 2.8μg/ml after 1hr, and the total recovery within 3hr from the urine was 30%. Saframycin A was found to be distributed widely, though to different extents, in various organs when injected intraperitoneally into mice.

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Skin fibroblasts from family members of cases of adenomatosis coli (AC) and Peutz-Jeghers' syndrome (PJ) were examined for susceptibility to morphological transformation by Moloney murine sarcoma virus (MSV) of rat 78Al cells, which was found in the present study to contain both dualtropic and ecotropic murine leukemia virus (MLV). The fibroblasts from individuals with manifest AC (22 subjects) and manifest PJ (5 subjects) were up to 5 times more susceptible to transformation by rat 78Al-MSV (MLV) than those from normal individuals (7 subjects). Nonmanifest members of AC families (8 subjects) showed various sensitivities: the fibroblasts from 3 family members had a sensitivity as low as that of normal cells, and those of 5 individuals exhibited increased sensitivity similar to that of manifest cases of AC. The AC and PJ cells that were sensitive to 78Al-MSV (MLV) were also susceptible to transformation by other pseudotypes of Moloney MSV, although they were not transformed by infection of standard Moloney MSV. None of the fibroblasts were transformed by xenotropic MLV alone. The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate enhanced transformation by 78Al-MSV (MLV) in both AC and normal cells.

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CD/CRJ rats were subjected to localized X-irradiation of the stomach and given N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the drinking water. Rats given MNNG alone and non-treated rats were used as controls. Upon sacrifice at 15 months after the initial MNNG administration, intestinal metaplasia was observed; the histology was of complete type and the incidence was 100% in rats treated with X-rays and MNNG, whereas in rats treated with MNNG alone the intestinal metaplasia was of incomplete type and its incidence was 80%. However, the incidence of gastric cancer in rats treated with MNNG alone was 25%.

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1-Butyl-1-nitrosourethan (BNUR) and 1-butylurethan (BUR), a precursor of BNUR, were administered prenatally or neonatally to ACI/N rats. A few neurogenic tumors were induced in the offspring of mother rats that had received BNUR at the late stage of pregnancy and in the animals that had received one subcutaneous injection of BNUR within 24hr after birth. No neurogenic tumors were observed in rats treated with BUR prenatally or neonatally.

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Cell surface antigens on neoplastic lymphoid cells of T cell lineage were analyzed with a fluorescence-activated cell sorter (FAGS-II) by utilizing rabbit antisera raised against human brain tissue (anti-Br) and fetal thymocytes (anti-Ty). Both before and after extensive absorption with normal peripheral blood lymphocytes (PBL), the anti-Br antiserum was capable of staining neoplastic lymphoid cells from patients with Sézary syndrome, mycosis fungoides, thymoma, and chronic lymphocytic leukemia. The absorption of anti-Ty with normal PBL, however, resulted in loss of the ability to stain these neoplastic cells. The anti-Br preabsorbed with normal PBL was found to stain normal bone marrow cells and neoplastic myeloid cells but not fetal thymocytes. It is suggested that the absorbed anti-Br is capable of detecting differentiation antigens present on immature hematopoietic cells in the bone marrow, which would be anomalously expressed on neoplastic lymphoid cells of T cell lineage.

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Transmission electron microscopic studies of precancerous and cancerous lesions in the pancreas of hamsters induced by N-nitrosobis(2-oxopropyl)amine (BOP) are presented. BOP was injected subcutaneously once weekly for 10 weeks and hamsters were sacrificed every 5 weeks after initiation of the experiment. The ultrastructural findings indicated that serial changes occurred in the epithelium of the pancreatic duct. The epithelial cells became cuboidal and showed increased secretions at 5 weeks. Probable precancerous cells with prominent nucleoli and irregular rough endoplasmic reticulum were found in the main duct at 10 weeks. At 15 weeks, pancreatic tumors forming a duct arrangement were seen, in good accord with the histological appearance. Well differentiated adenocarcinoma cells showing a tubular pattern had oval nuclei with granular chromatin. Poorly developed rough endoplasmic reticulum was irregularly distributed throughout the cytoplasm and the cell surface was covered with microvilli. Poorly differentiated adenocarcinoma showed poor gland formation and had distorted nuclei with prominent nucleoli. These cells were loosely joined. Mitochondria and rough endoplasmic reticulum were poorly developed, and the tumor cells were devoid of secretory granules.
The most characteristic and common change of the precancerous and cancerous lesions in this experiment was the appearance of numerous microvilli on the luminal surface and loss of cytodifferentiation. These findings were obviously different from those of normal epithelial cells or those seen in inflammation. The findings in this study confirm that the pancreatic carcinoma induced by N-nitrosobis(2-oxopropyl)amine in Syrian hamsters is of duct cell origin. No evidence of acinar cells was obtained.

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The effects of various hepatocarcinogenic, non-hepatocarcinogenic and noncarcinogenic chemicals on the induction of hyperplastic liver nodules by N-nitrosodiethylamine (DEN) or N-2-fluorenylacetamide (2-FAA) as an initiator were studied in male Fischer 344 rats. Rats were injected intraperitoneally with 200mg of DEN/kg body weight or were fed on basal diet containing 200ppm of 2-FAA for 2 weeks, and then given various test chemicals starting from week 3. They were also partially hepatectomized in week 3. All animals were killed at the end of week 8 and examined histologically. For quantitative analysis, hyperplastic nodules in the liver were measured with a color video image processor, VIP-21C. The effects of various chemicals in rats treated with DEN or with 2-FAA were compared. The production of hyperplastic liver nodules was greatest in rats treated with strong hepatocarcinogens, and less in rats treated with weak hepatocarcinogens. Very few hyperplastic nodules were produced after treatment with non-hepatocarcinogens or noncarcinogens. Hyperplastic nodules were formed in rats treated with phenobarbital, which is a hepatopromoter. Saccharin, which is a urinary bladder promoter, did not enhance the production of hyperplastic nodules in the liver. These results indicate that many hepatocarcinogens enhance liver carcinogenesis. The classification of chemicals as liver carcinogens is discussed on the basis of the results.

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The behavior of hyperplastic nodules following an in vivo short-term screening test for hepatocarcinogens was studied. Rats were injected ip with 200mg/kg body weight of diethylnitrosamine (DEN), given basal diet containing 200ppm of N-2-fluorenylacetamide (2-FAA) (group 1), 1000ppm of the α-isomer of 1, 2, 3, 4, 5, 6-hexachlorocyclohexane (α-BHC) (group 2) or basal diet (group 3) from week 3 to week 8, and then given basal diet and tap water. They were subjected to partial hepatectomy at the end of week 3. Animals were killed at weeks 4, 6, 8, 10, 20, 30, 40, and 50. A significant disappearance of hyperplastic nodules following the cessation of carcinogen treatment was observed in group 1, but was not evident in groups 2 and 3. With γ-glutamyltranspeptidase (GGTase) as a positive marker and adenosine triphosphatase (ATPase) as a negative marker, hyperplastic nodules were classified into 3 different phenotypic categories, i.e., (1) GGTase-positive and ATPase-negative, (2) GGTase-positive, and (3) ATPase-negative. The percentages of GGTase-positive and ATPase-negative hyperplastic nodules were about 80∼90% in group 1 and 70∼80% in groups 2 and 3. Some of the hyperplastic nodules were necrotic from week 8 in groups 1 and 2, and from week 20 in group 3. Subsequently, the numbers of necrotic hyperplastic nodules increased with time. Hepatocellular carcinomas were found at weeks 30, 40, and 50 in group 1, and at weeks 40 and 50 in group 2. Significantly higher incidences of cancer were found in group 1 than in group 2. The hepatocellular carcinomas were also classified enzyme-histochemically into 3 different phenotypic categories as for hyperplastic nodules, but the percentage (20%) of GGTase-positive and ATPase-negative hepatocellular carcinomas was significantly lower than that (70∼90%) of GGTase-positive and ATPase-negative hyperplastic nodules in each group.

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The metabolic fate of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was studied in the rat, to investigate the possibility of a relationship between urinary metabolites and organotropic carcinogenicity to the urinary bladder of this N-nitrosamine. The principal urinary metabolite of BBN was identified as N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN). Several minor metabolites characterized were transformation products of BCPN formed by β-oxidation according to the Knoop mechanism, i.e., N-butyl-N-(2-hydroxy-3-carboxypropyl)nitrosamine, N-butyl-N-(carboxymethyl)nitrosamine and N-butyl-N-(2-oxopropyl)nitrosamine; glucuronic acid conjugates of BBN and BCPN were also detected. No BBN was detected in the urine. A possible correlation of the urinary excretion of BCPN with selective induction of bladder tumors by BBN in rats is discussed in relation to the carcinogenic action of BCPN.

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The metabolic fate of N, N-dibutylnitrosamine (DBN) was studied in the rat, to elucidate the possibility of a correlation between its metabolism and its organotropic carcinogenicity to the urinary bladder and other organs. It was extensively metabolized in the rat, no unchanged DBN being found in the urine. DBN underwent metabolic transformation in at least three ways. The major pathways demonstrated on the basis of urinary metabolites were ω- and (ω-1)-oxidations of one butyl chain to give N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN) and N-butyl-N-(3-hydroxybutyl)nitrosamine, respectively. The third minor pathway was (ω-2)-oxidation of the butyl chain to afford N-butyl-N-(2-hydroxybutyl)nitrosamine. Both hydroxylated metabolites were excreted into the urine as such and as their glucuronic acid conjugates. The ω-oxidation of DBN to BCPN is responsible for the induction of bladder tumors in rats, while the products of the (ω-1)- or (ω-2)-oxidation may be involved in tumor induction in the liver.

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The growth of hamster embryonic fibroblasts was highly dependent on the serum concentration in the medium. A tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), enhanced the growth of hamster embryonic fibroblasts in the medium containing low concentrations of serum. Among phorbol derivatives, phorbol-12, 13-didecanoate and 4-O-methyl-12-O-tetradecanoyl-phorbol-13-acetate also showed growth stimulation, but 4α-phorbol-12, 13-didecanoate, phorbol-12, 13-diacetate and phorbol did not. TPA increased the accumulation of 2-deoxyglucose into the cells at low serum concentrations. Thus, hamster embryonic fibroblasts were sensitive to phorbol esters as growth stimulators, and TPA reduced the serum requirement of the cells.

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Changes in the glycogen phosphorylase isozyme patterns of AH130 and AH66F during cell growth were investigated immunochemically and electrophoretically. The content of the liver-like type increased as the cell growth in the ascites decreased, while the level of the fetal type (or prototype) remained approximately constant. The isozyme shift from the fetal type to liver-like type was more marked in subcutaneous solid AH130. In AH130 cells cultured in vitro under growth-inhibited conditions, the content of the hybrid between the above two types was increased, with increased amount of liver-like subunit. These results indicate that the phosphorylase isozyme patterns of Yoshida ascites hepatomas vary during cell growth under different conditions.

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Xenoantiserum to human malignant melanoma was prepared by immunizing rabbits with melanoma associated antigens (MAA) solubilized from melanoma cell membrane by limited papain digestion. The antiserum was absorbed extensively with red cells, leukemia cells and cultured lymphoid cell lines, and was assayed for its reactivity with different human cell types by the indirect membrane immunofluorescence and radioimmunoprecipitation techniques. The data obtained suggest that there are at least two different MAA on human melanoma cells. The first is melanoma-group specific and can be detected commonly on different melanoma cell lines. The second is oncofetal and is shared by melanoma, carcinoma and fetal cells tested thus far. Immunoprecipitated material from melanoma cell membrane that had been radioiodinated with lactoperoxidase and solubilized with a non-ionic detergent was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed two major peaks with estimated molecular weights of 90, 000 and 120, 000 daltons. The 90, 000 molecular weight component appears to be oncofetal, as it disappeared when the antiserum was absorbed with either melanoma or carcinoma cell lines.

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The metabolism of 1-(2-tetrahydrofuryl)-5-fluorouracil (FT) plus uracil (1:4, UFT) was compared with that of FT alone and that of uracil alone in tumor-bearing rats by using ³H-tracer.
After oral administration of UFT, both FT and uracil were rapidly absorbed and no interaction of their absorptions was observed. The levels of 5-fluorouracil (5-FU) in the blood and tissues and the excretion of 5-FU in the urine of rats given UFT were temporarily higher, and the levels of α-fluoro-β-ureidopropionic acid (a catabolic metabolite of 5-FU) in almost all tissues were lower than in rats given FT.
The levels of 5-FU, 5-fluorouridine, and fluoronucleotides increased more in the tumors than in normal tissues of rats given UFT, but not in those given FT.
The metabolism of uracil administered exogenously as UFT was not influenced by 5-FU or its metabolites derived from FT.

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A pyranyl derivative was chemically synthesized from secalonic acid D, an antibiotic obtained from culture filtrates of Penicillium oxalium, and its anticancer activity towards a highly antigenic rat bladder cancer, BC-47, implanted into inbred ACI/N rat was studied. The anticancer effect of the drug was similar to that of adriamycin. Delayed initiation of treatment, starting 5 days after the cancer implantation, was more effective than treatment starting from day 1. In addition, it was less effective in an immunodeficient host subsequently implanted with BC-47. This compound is thought to retard the cancer cell growth until the appearance of tumor immunity in the host.

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An intravenous injection of 5×10⁶ washed, viable Ehrlich ascites tumour cells given to mice 2hr before, immediately before, or 2hr after a dose of 60μg/g body weight of cycloheximide, results in such animals being either dead or moribund 48hr later. No fatalities occur when a similar number of tumour cells are given 2hr before or 2hr after a dose of 30μg/g body weight of cycloheximide. Similarly, 5×10⁶ tumour cells are well tolerated when given to mice 18hr after 60μg/g cycloheximide. It is suggested that an undefined minimum level of recipient protein synthesis is required for maintenance of life in the presence of a challenge by 5×10⁶ tumour cells. A minimum period (>6hr) is needed for an irreversible illness to result from interaction of tumour cells with a recipent animal under the maximal influence of the 60μg/g dose of cycloheximide. The irreversible illness is not avoided by the use of tumour cells pretreated for 2hr with 30μg/g of cycloheximide, nor is the outcome altered when both recipient and tumour cells are together subjected to a 60μg/g dose of cycloheximide.

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A potent hepatocarcinogen, diethylnitrosamine (DEN), was orally administered at 50ppm for 5 weeks to Wistar rats preexposed to polychlorinated biphenyls (PCB) in utero and via mother's milk. The numbers of liver tumors induced by administration of DEN were significantly reduced in the rats exposed to PCB in an early stage of life, compared with control rats. This tumor-inhibiting action was particularly clear in male offspring.

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The effect of leupeptin, a protease inhibitor, on the development of spontaneous tumors was studied in strain A mice. Three-week-old mice were divided into two groups: one group was fed on diet containing 0.1% leupeptin and the other was fed on basal diet. The experiment was terminated 480 days after the start of administration of the leupeptin diet. Almost all animals survived until the end of the experiment. In the group given the leupeptin diet, 15 of 50 mice developed liver tumors (14 of 25 males and 1 of 25 females). In the control group, only male mice developed liver tumors (4 of 23 males). There was a significant difference between the incidences of liver tumors in males fed leupeptin diet and basal diet, but there was not in the case of females.

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