The effect of potassium bromate, a food additive, was examined in male F-344 rats based on two-stage renal carcinogenesis with N-ethyl-N-hydroxyethylnitrosamine (EHEN) as an initiator. Average numbers of dysplastic foci and renal cell tumors were significantly increased in rats given potassium bromate after initiation with EHEN. It was concluded that potassium bromate has an enhancing effect on renal tumorigenesis.

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1α, 25 -Dihydroxyvitamin D₃, a hormonally active form of vitamin D₃, enhanced methylcho-lanthrene-induced transformation in BALB 3T3 cells to a much greater extent than 12-O-tetradecanoylphorbol-13-acetate. This enhancement was probably mediated by a cytosol receptor for 1α, 25-dihydroxyvitamin D₃ which has an equilibrium constant of 28.4pM and a maximum binding of 32.6fmol/mg protein.

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Human fetal lung tissue was cultured on dead, sterile pigskin as a substrate and treated with 3-methylcholanthrene. Several cell lines were obtained. One of the lines produced a tumor in nude mouse and showed anchorage-independent growth in soft agar. The tumor cells were stained positive for keratin, indicating their epithelial origin.

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P388 leukemia cells resistant to vincristine contained more calcium in the cells than the parent sensitive line, especially in the form of EGTA-removable (surface-bound) calcium. Although the relationship, if any, between the high cellular calcium of resistant cells and drug-resistant phenotype is not clear, the possible implications of this result for the elucidation of the mechanisms of drug resistance are interesting.

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As hyperthermia treatment for deeply located malignant lesions is most difficult, two types of endotract antenna were devised, one constructed for radio frequency and the other for administration of microwave to tumors of the esophagus and colorectum. Well-controlled temperatures can be applied to the narrowed pyloric canal of dogs with these two instruments, which appear to have great promise for clinical application.

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The effects of sodium o-phenylphenate (OPP-Na) and o-phenylphenol (OPP) on 2-stage urinary bladder carcinogenesis in F344 rats initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) at levels of 0.01 or 0.05% in drinking water were investigated. Administration of 2.0% OPP-Na in the diet significantly increased both the incidence and the number per 10cm of basement membrane of preneoplastic lesions (papillary or nodular hyperplasia; PN hyperplasia) of the urinary bladder in rats pretreated with 0.01% BBN, and also those of papilloma and cancer of the urinary bladder in the group pretreated with 0.05% BBN. Moreover, treatment with 2.0% OPP-Na, without prior BBN initiation, induced PN hyperplasia, papilloma and cancer. In contrast, OPP appeared to produce only a slight increase in the incidence of urinary bladder lesions following BBN, and its effect was not statistically significant. It also did not induce tumors of the urinary bladder. These results provide evidence that OPP-Na has promoting activity toward the urinary bladder while OPP does not. Moreover, the possibility that OPP-Na may be a complete carcinogen in the rat urinary bladder deserves consideration.

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The carcinogenicity of fish meal pyrolysate was examined in noninbred Syrian golden hamsters of both sexes. Hamsters were given a diet containing 5%, 10%, 20% or 40% fish meal pyrolysate for 102 weeks in Experiment 1, and 10% or 20% fish meal pyrolysate for 112 weeks in Experiment 2. Hamsters in control groups in both experiments were fed a normal basal diet. Various tumors were found in both experimental and corresponding control groups, but there was no significant difference in the incidence of any tumors between the experimental and control groups. Thus, the fish meal pyrolysate tested was not carcinogenic to Syrian golden hamsters under these conditions. In male hamsters the severity and incidence of fatty degeneration of the liver and chronic nephropathy tended to correlate with the concentration of fish meal pyrolysate in the diet. With females, however, the correlation was not so clear. In neither sex did the presence of atrial thrombosis or systemic amyloidosis appear to be dependent on the dietary concentration of fish meal pyrolysate.

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To examine the possible carcinogenic effects of frequent intake of mutagenic pyrolysates of proteins and amino acids, 7, 553 adult subjects whose personal characteristics had been examined were followed-up for about 11 years. The stepwise multiple regression analysis revealed that frequent consumption of broiled fish was significantly positively associated with mortality from cancer at all sites, as well as from cancer of the stomach. Among the 11 independent variables tested, consumption of broiled fish ranked high as a variable associated with cancer mortality (all sites, and stomach). Similar analyses done by sex and city (Hiroshima and Nagasaki) showed a consistently positive association of this variable with mortality from cancer at all sites, in both sexes and cities, but, with a few exceptions, the association was not statistically significant. Broiled fish consumption also showed a consistently positive but not statistically significant association with mortality from gastric cancer classified by sex and city. The relative risks of cancer mortality associated with frequent (twice or more weekly) consumption of broiled fish, as compared to less frequent consumption, were 1.3 (P<0.05) for cancer at all sites and 1.7 (P<0.05) for gastric cancer. The frequency of consumption of dried fish was hardly associated with deaths from cancer at all sites and from gastric cancer, but was significantly positively associated with deaths from cancer of the liver.

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Changes in the activities of rat hepatic glutathione (GSH) peroxidase (GSH-Px) and GSH reductase (GR) were investigated during the induction of preneoplastic γ-glutamyltransferase (γ-GT)-positive foci and hyperplastic nodules by the administration of diethylnitrosamine followed by N-2-fluorenylacetamide. Activities of GSH-Px towards both cumene hydroperoxide (cumene-OOH) and hydrogen peroxide markedly decreased at an early stage of the above hepatocarcinogenesis, but the activity towards cumene-OOH again increased with the appearance of the foci and nodules, which were detectable as an increased γ-GT activity. It was demonstrated by CM-Sephadex column chromatography and immunochemically that the increased activity towards cumene-OOH alone is due to the increased GSH-Px activity of GSH S-transferase B. GR activity and the total glutathione content in the liver also increased with increased preneoplastic foci and nodules. These results are discussed in connection with the protection mechanism(s) of these cell populations against damage by active oxygen and lipid peroxides produced in chemical hepatocarcinogenesis.

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In rats bearing a solid form of AH-109A hepatoma, serum asialofetuin sialyltransferase activity was significantly increased. In order to identify the source of the increased serum sialyltransferase, the asialofetuin sialyltransferase activities of normal and host liver, tumor, and normal and host serum were studied by phosphocellulose column chromatography. While normal and host (day 17) livers exhibited two peaks, namely, transferases I and II, which were previously shown to be the sialylated and unsialylated species, respectively, of β-galactoside α2→6 sialyltransferase, the tumor exhibited a single peak of the enzyme which was a sialylated species (as was transferase I) but was eluted at a position clearly distinguishable from that of either transferase I or transferase II. Under these conditions, both normal and host (day 17) sera were found to contain transferase I but not the tumor-type enzyme. The results have been interpreted as indicating that in rats with AH-109A, the tumor is not the source of the increased serum sialyltransferase. In these rats as well as in normal rats, serum sialyltransferase appears to originate mainly from the liver, whose sialyltransferase activity was also increased in rats with AH-109A.

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Dose-related suppression of the ³H-thymidine incorporation into liver DNA of rats after a single injection of dimethylnitrosamine by copper pretreatment was observed. The ³H-thymidine incorporation was not decreased by cadmium pretreatment. On the other hand, the ³H-thymidine incorporation into liver DNA of partially hepatectomized rats was decreased by both copper and cadmium pretreatments. Thymidine kinase activity in the liver of rats treated with dimethylnitrosamine was also decreased by copper pretreatment, but the enzyme activity was not decreased by cadmium pretreatment. Copper accumulation in the liver of copper-administered rats was predominantly in the nuclear fraction, followed by the soluble fraction. Cadmium accumulation in the liver of cadmium-administered rats was predominantly in the soluble fraction, followed by the nuclear fraction. Copper accumulation in the nuclear fraction may suppress the induction of thymidine kinase in the liver of rats by dimethylnitrosamine.

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Media conditioned with 8 cell lines were assayed for stimulating and inhibiting activities on granulocyte-macrophage colony formation from bone marrow cells in soft agar (CFUc). Conditioned media from 3 human bladder cancer cell lines (EJ, T24 and KK47) and a rat bladder cancer cell line (BC50-TC) showed marked inhibition of mouse CFUc. Conditioned medium from normal human bladder cells showed a weak colony-stimulating activity, and only a slight inhibition when added to CFUc culture. Conditioned media from mouse or rat fibroblast cell lines (BLP and 3Y1) showed no colony-inhibiting activity, and the conditioned media from human lymphoblastoid cell line (Raji) and human epithelia-like cell line from amniotic membrane (FL) showed moderate inhibitory activity. No colony-stimulating activity was detected in any of the cell lines tested. EJ-conditioned medium inhibited human CFUc, but not erythroid progenitors. Colony-inhibiting activity in EJ-conditioned medium appears not to be prostaglandins; it needs more than 3hr to inhibit CFUc, is stable at 56° for 30min but not at 75° for 20min, and is susceptible to proteolytic enzymes.

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A quantitative study of carcinoembryonic antigen (CEA) was made in nine human gastric cancer cell lines. Six of them were found to produce CEA in vitro. The production of CEA in the three cell lines derived from well differentiated tubular adenocarcinomas began at the mid-exponential phase of cell growth and reached its peak at the late stationary phase, the amount of CEA per 10⁵ cells and the frequency of CEA-positive cells on immunostaining increased with culture time. In contrast, CEA in the three cell lines derived from poorly differentiated adenocarcinomas including a signet-ring cell carcinoma was produced immediately after plating and the amount of the antigen per 10⁵ cells and the frequency of CEA-positive cells were almost constant throughout the cell growth phases. Serum CEA content in nude mice was low or not detectable in the case of subcutaneous heterotransplantation of gastric cancer cells, irrespective of CEA productivity of the cell lines in vitro. Intraperitoneal inoculation, however, led to high CEA levels in sera of nude mice bearing human gastric cancers. No significant difference was found between the two kinds of inoculation in terms of the total tumor weight and the frequency of CEA-positive cells in tumor tissues. One reason for the above findings may be that the transport of CEA in the subcutaneous tumors to the systemic blood flow is hindered.

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The effects of laminin, fibronectin and type IV collagen, all of which are major matrix components of the basement membrane, upon fetal mouse hepatocyte culture were studied. Among these matrices, laminin showed the greatest effect on DNA synthesis, cell proliferation, the secretion of α-fetoprotein and cell attachment. The effects of fibronectin and type IV collagen were slight with regard to the promotion of growth and cell attachment. However, there were no differences in the secretion of albumin into the media among the groups. These results suggest that laminin may be necessary for the cellular growth and functional maintenance of immature liver cells during normal development of the liver in vivo.

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Misonidazole, a derivative of 2-nitroimidazole, has selective cytotoxic activity on hypoxic cells in addition to its radiosensitizing activity. This cytotoxicity is considered to be due to metabolic reduction of the drug. A possible metabolite seems to be hydroxylaminomisonidazole, an intermediate product derived via reduction of the nitro group. Authentic samples of hydroxylamino- and aminomisonidazole (a final reduction product) were synthesized and their cytotoxicity towards HeLa S3 cells was compared with that of misonidazole. After a 3-hr exposure to 1mM hydroxylaminomisonidazole under aerobic and hypoxic conditions, the surviving cell fractions were 0.18 and 0.0056, respectively. This represents a cytotoxicity five and 125 times greater, respectively, than that of misonidazole. Under the same conditions, aminonusonidazole showed no apparent cytotoxicity.

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The survival of cultured mouse L cells and the progression of the cells through the cell cycle after exposure to aclacinomycin-A (ACM-A) were studied. At low drug concentrations, there was a slight depression in survival of S phase cells, while at high concentrations, cells at late G₁ and late S-G₂ were very sensitive to the drug. The dose-survival curve of synchronous cells was similar to that of asynchronous cells. Initially, there was a small reduction in survival, but as the dose was increased no additional killing occurred until a certain level was exceeded, after which an exponential decline in survival resulted. The age-response with high concentrations of the drug reflected the differences in the size of the shoulder of the dose-response curve, while that with low drug concentrations reflected the presence or absence of the initial small decrease in survival. ACM-A inhibition of cell progression was greatest for cells in mid G₁ followed by cells in late S and finally by cells in G₂.

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The eyes of 10 F344 rats were inoculated with retinal tumor cells (EXP-5 cell line) induced by human adenovirus 12. The animals were killed at 4 weeks thereafter, and the cytotoxicity of their lymphocytes was investigated by using ⁵¹Cr-releasing assay. The percentage of EXP-5 cells killed in vitro by lymphocytes was higher in 10 rats with ocular tumors (24.6%±6.1%, mean±SD) than in 10 control rats (6.2% ±1.8%). Morphologic investigation using syngeneic spleen cells confirmed the presence of lymphoid cells, resembling T-lymphocytes, adhering to EXP-5 cells. The influence of subcutaneous injection of EXP-5 cells on the growth of intravitreously injected tumor cells was investigated. Cells injected subcutaneously prior to intravitreous injection elicited an immune response that was capable of controlling vitreous tumor growth. These findings suggest that the rats with transplanted retinal tumors develop a cell-mediated immune response in the early stage of tumor bearing, and that a state of pre-existing specific immunity can overcome so-called “immunologic privilege” of the vitreous body.

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A considerable number of normal mice injected with a low dose of A/Ph.MC.S1 syngeneic methylcholanthrene-induced tumor cells showed regression of the tumor after a transient period of growth. The regressor mice eliminated a challenge inoculum in an accelerated fashion. Splenic lymphocytes from such regressor mice inhibited the growth of the same tumor in the local adoptive transfer assay. This capacity required the presence of thymus-derived cells. The regressor animals developed a delayed-type hypersensitivity response against the tumor In vivo and their lymph-node cells produced macrophage migration inhibitory factor in the presence of tumor cells in vitro. The growth of this tumor was facilitated by treating the primary recipients with carrageenan, and the strong tumor-inhibitory capacity of regressors was also depressed by this agent. Early macrophage infiltration of the tumor in regressor mice was demonstrable histochemically, preceding the inhibition of tumor growth. A lymphokine-producing T cell-mediated, macrophage-dependent delayed-type hypersensitivity-like mechanism is proposed to be the dominant mechanism of the resistance in this tumor-host model.

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An ovarian cancer patient was treated with human fibroblast interferon (HFIF) given daily by iv infusion, and the lymphocyte natural killer (NK) activity against K562 and the cytotoxicity against autologous tumor cells under various experimental conditions were investigated. After HFIF treatment, NK activity against K562 increased significantly, whereas the autologous tumor cell kill by lymphocytes was not increased with target tumor cells that were fresh, frozen then thawed, or frozen, thawed and cultured for 4 to 5 days before use. In vitro HFIF-stimulated lymphocytes also did not enhance cytotoxicity against the various autologous tumor cells. However, lymphocytes from allogeneic healthy persons did show enhanced cytotoxicity against cultured tumor cells of this patient after cultivation with HFIF, but had no significant effect against frozen tumor cells. Mixed lymphocyte-tumor cell reaction (MLTR) and MLTR-induced lymphocyte cytotoxicity against autologous tumor cells were not enhanced by HFIF. Thus, tumor-specific immune reactions directed toward autologous tumor cells were not enhanced by HFIF treatment under the conditions tested in this study.

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Mouse interferon (MuIFN) strongly suppressed the growth of human nasopharynx carcinoma (KB) transplanted into nude mice when given by subcutaneous injection around the tumor, though KB cells were insensitive not only to the antiproliferative activity of MuIFN, but also to natural killer (NK) activity in vitro. Moreover, the antitumor effect of MuIFN was not influenced by treatment with anti-asialo GM1, which eliminates NK activity in vivo. These results indicate that NK activity is not essential to the antitumor effect of IFN mediated by the host. Subcutaneous injection of MuIFN was more efficacious than intraperitoneal injection, suggesting the existence of regional tumor resistance of the host induced by IFN. The sera of mice treated with MuIFN showed no inhibitory activity on the growth of KB cells in vitro, indicating that no antiproliferative agent secondarily induced by IFN treatment is present in the serum. There was no difference between control and MuIFN-treated group in terms of the histology of tumor tissues in nude mice. An undefined host-dependent antitumor mechanism distinct from the proposed immune modulatory effect of IFN might be present.

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The growth-inhibitory activity of recombinant human interferon-β (ReIFN-β) against cultured human cells was compared with that of natural human fibroblast interferon-β (IFN-β), and the influence of deficiency of carbohydrate on the unicellular activity was examined. The IC₅₀ (concentration of drug required for 50% inhibition) of ReIFN-β against 14 human cell lines was almost equivalent to that of IFN-β, when the cells were cultured for 7 days and ReIFN-β or IFN-β was added on day 0 and exchanged every day from day 1 to day 6. The most sensitive cells (ICE<10units/ml) were Daudi lymphoma cells and 3 melanoma cell lines, and the most insensitive cells (IC₅₀>10³units/ml) were HeLa S3/IS cells (insensitive line) and CCRF-CEM leukemia cells. The other 8 cell lines were moderately sensitive to both interferons. As the intervals of exchange of ReIFN-β or IFN-β were extended, the growth-inhibitory activity of both interferons decreased. This phenomenon, which was more significant with ReIFN-β than IFN-β, was explicable in terms of the stability of both interferons incubated in the culture medium at 37°. The species specificity of IFN-β was not mediated by carbohydrate since the growth-inhibitory activity of ReIFN-β against 2 mouse cell lines was almost equivalent to that of IFN-β. These results indicate that the anticellular activity of ReIFN-β was not essentially affected by deficiency of carbohydrate.

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Highly purified recombinant fibroblast interferon produced by Escherichia coli (ReIFN-β) was tested for its ability to stimulate natural killer (NK) cell activity, antibody-dependent cell-mediated cytotoxicity (ADCC) and monocyte-macrophage phagocytic activity in human peripheral blood mononuclear cells (PBM) in comparison with natural human fibroblast interferon (IFN-β). The NK cell activity against target cells (K-562, Molt-3, CCRF-HSB-2, CCRF-CEM, Daudi, and HeLa S3) was enhanced by ReIFN-β and IFN-β. Augmentation of cytotoxicity of NK cells was observed at a concentration of ReIFN-β as low as 1U/ml and increased in a dose-dependent manner. The enhancing effect of ReIFN-β was expressed sufficiently during a 1-hr incubation with effector cells, and IFN-β showed kinetics and potency similar to those of ReIFN-β. ADCC was measured with chicken red blood cells (CRBC) or CCRF-CEM coated with each antibody, as target cells. Pretreatment of PBM with ReIFN-β caused a significant increase in ADCC activity against both targets, and the enhancing activity of ReIFN-β was similar to that of IFN-β. Phagocytic activity of adherent cells in PBM against CRBC was enhanced by ReIFN-β and IFN-β as determined by microscopical cytologic examination and by the ⁵¹Cr-labeled CRBC-uptake method. From these results, it can be concluded that recombinant human IFN-β modulates NK cell activity, ADCC and phagocytic activiity of human PBM as effectively as natural human IFN-β.

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A vincristine (VCR)-resistant subline of human K562 myelogenous leukemia was established in vitro, and several clones with different susceptibilities to VCR were isolated by the limiting dilution technique. The most resistant clone (H-1) had a 17-fold greater resistance to VCR when compared to the parent K562 cells. The clone gradually lost the resistance during prolonged culture in vitro. These clones generally accumulated smaller amounts of VCR in their cells as compared to the parent cells. The size of H-1 clone cells was almost the same as that of the parent cells. The numbers of potential binding sites of VCR in the K562 cells and the resistant H-1 clone were almost the same. Similar results were obtained for P388 and its VCR-resistant sublime. The cells derived from the VCR-resistant H-1 clone were highly cross-resistant to vindesine and moderately resistant to vinblastine. Cells derived from clone H-1 exhibited marginal degrees of cross-resistance to adriamycin, maytansine and VP-16-213, whereas VCR-resistant P388 leukemia cells exhibited significant resistance to these agents, especially to maytansie.

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Mycotrienins I and II and mycotrienols I and II are ansamycin antibiotics containing a triene structure, isolated from Streptomyces rishiriensis. An amphotericin B-resistant clone (AMB^R-1) derived from cultured Chinese hamster V79 cells was cross-resistant to mycotrienins I and II, but not to mycotrienol I or II. These four ansamycin antibiotics were found to potentiate significantly the action of some anticancer agents including 5-fluorouracil, cis-diamminedichloroplatinum, bleomycin, mitomycin C and 6-mercaptopurine against cultured V79 cells. The action of adriamycin was not potentiated. These four ansamycin antibiotics showed a synergism spectrum similar to that of smaller polyene antibiotics.

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The cytotoxic effect of peplomycin (PEP), a new derivative of bleomycin glycopeptide antibiotics, toward HeLa cells and mouse FM3A cells was markedly enhanced by combination of PEP with non-toxic doses of the membrane-interacting drugs verapamil (0.1-0.2mM) and dibucaine (0.15-0.25mM), or with CaCl₂ (10-16mM). Treatment with verapamil or CaCl₂ following PEP treatment also effectively enhanced the cytotoxic effect of PEP, suggesting an interaction with PEP-induced damage. Cellular uptake of PEP did not increase in dibucaine-treated cells, suggesting no correlation between membrane permeability to PEP and enhanced cytotoxicity. Increased Ca²⁺ did not enhance the cytotoxic effects of adriamycin, mitomycin C, cis-diamminedichloroplatinum (II), vinblastine or macromomycin, thus suggesting a unique interaction with PEP. The enhancing action of verapamil and Ca²⁺ was greatly promoted by 41° hyperthermia, although 41° alone scarcely enhanced PEP cytotoxicity. Cytochalasin B and α-tocopherol, but not cytochalasin D, reversed the enhanced cytotoxicity produced by PEP and verapamil or dibucaine, but did not reverse the enhanced cytotoxicity by PEP combined with increased Ca²⁺. The enhancing action of Ca²⁺ was, however, antagonized by ruthenium red and lanthanum chloride, which are potent inhibitors of Ca²⁺ uptake by cells, or by magnesium chloride. Verapamil and increased Ca²⁺ promoted the decomposition of the DNA-membrane complex induced by PEP in HeLa cells, and also impaired the regeneration of the decomposed DNA complex.

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