The in vitro metabolism of the antitumor anthracycline antibiotic, aclacinomycin-A, was studied using rat liver homogenate. In the presence of NADH or NADPH, aclacinomycin-A was converted to aclacinomycin-A analogs, MA144 Ml and MA144 Nl, which were stereospecifically reduced at the keto group of the C-4''' position of L-cinerulose in aclacinomycin-A. Subcellular fractionation indicated that the production of MA144 Ml, which was reduced to L-amicetose, was catalyzed by NADPH-dependent soluble cinerulose reductase I, and the production of MA144 Nl, which was reduced to L-rhodinose, was catalyzed by NADPH-dependent soluble cinerulose reductase II and NADH-dependent microsomal cinerulose reductase. The properties of these three enzymes were studied. Soluble cinerulose reductase I which produces MA144 Ml showed an optimum pH at 6.3, K_m values of 3.3×10^-4M for aclacinomycin-A and 3.2×10^-5M for NADPH. Soluble cinerulose reductase II which produces MA144 Nl Showed a pH optimum at 6.3 and K_m values of 2.0×10^-3M for aclacinomycin-A and 4.0×10^-5M for NADPH. All these reductases were sensitive to sulfhydryl reagents and were inhibited by vitamin K₃. Microsomal cinerulose reductase showed sensitivity to dicoumarol and ferrous ion. The main nondegradative pathways of aclacinomycin-A were discussed from these results.

View full abstract

The in vitro degradation of the new antitumor anthracycline antibiotic, aclacinomycin-A, was studied using rat liver homogenate. In the presence of NADH or NADPH, the glycosidic bond at C-7 position of aclacinomycin-A was reductively cleaved to produce 7-deoxyaklavinone and 7-deoxyaklavinone dimer, MA144 El. Subcellular fractionation indicated that most of the enzyme activity was present in the microsomal fraction and required anaerobic condition and NADPH. The purified enzyme reduced the glycosidic metabolites, MA144 Ml and MAl44 Nl, as well as aclacinomycin-A. The optimum pH for the anthracycline glycoside reductase reaction using aclacinomycin-A as substrate was 7.4. The enzyme was sigmoidally saturated with aclacinomycin-A and showed the concentration of 1.2×10^-4M required for half maximal activity, and K_m value of 7.7×10^-5M for NADPH. The degradative pathway of aclacinomycin-A and its glycosidic metabolites was discussed.

View full abstract

Aclacinomycin-A is a new anthracycline glycoside and has less cardiotoxicity than adriamycin. In an attempt to provide an experimental model of a phase III study of aclacinomycin-A, particularly for the treatment of malignant lymphomas, various therapeutic designs of combinations of this drug with other conventional agents were investigated using a P388 mouse leukemia system. Aclacinomycin-A showed no treatment schedule dependency in this tumor system and the optimal dosage of this drug was twice higher than that of adriamycin on each treatment schedule; i. e., single treatment on day 1, three treatments on days 1, 5, and 9, or 10 treatments on every other day from days 1 to 19 after an inoculation of 10⁶ leukemic cells on day 0. This antibiotic was ineffective against an adriamycin-resistant subline of P388 leukemia. Among combinations of aclacinomycin-A with cyclophosphamide, vincristine, procarbazine, or bleomycin, the combinations of aclacinomycin-A with cyclophosphamide or vincristine showed a therapeutic synergism in P388 leukemia system.

View full abstract

Mice sensitized with either BCG or cell-wall skeleton of BCG (BCG-CWS) produced interferon in blood after stimulation with specific antigen, purified protein derivative of tuberculin (PPD). Both BCG-infected normal (C57BL/6) and athymic nude (BALB/c, nu/nu) mice showed enhanced activity to produce interferon by stimulation with E. coli endotoxin. However, detectable interferon was not produced in athymic nude mice sensitized with BCG or BCG-CWS by stimulation with PPD. Immune-induced interferon (I-IF) produced by BCG-CWS and PPD in mice was different in biological and physicochemical properties from virus-induced interferon. I-IF showed about 100 times more potent L-cell growth inhibitory activity than virus-induced L-cell interferon (L-IF). Both I-IF and L-IF showed macrophage-activating activity, which renders resting macrophages cytotoxic to L1210 leukemia cells. Antiviral and macrophage-activating activity of interferon preparation was not separated physicochemically in this study.

View full abstract

Antitumor activity of marine bacteria, Vibrio anguillarum P-B-1, against Ehrlich carcinoma cells in ddY mice was investigated. Ehrlich carcinoma cells were inoculated intraperitoneally (ip) into mice and V. anguillarum (1.0mg/mouse) was administered ip twice before and 4 times after the tumor inoculation. Groups of mice administered V. anguillarum survived 80∼90% and their mean survival was 54.0∼56.0 days (range, 24.0∼60 days) at day 60 after inoculation of 10⁴ to 10⁶ tumor cells against the mean survival of 16.4∼22.3 days (range, 7∼29) in the control group. When 2×10⁶ tumor cells were inoculated subcutaneously (sc) mixed with 1.0mg of V. anguillarum, the bacterial cells markedly suppressed the growth of tumor at the injection site; tumor did not grow in 30% of recipient mice and the inhibition rate of grown tumor in the rest of recipients was 69%.
The consistently demonstrable antitumor activity of V. anguillarum was reduced by pretreatment of mice with immunosuppressants such as anti-thymocyte serum, hydrocortisone, or irradiation of X-ray. This fact indicates that the antitumor activity of V. anguillarum is mediated by immune response.

View full abstract

A study was undertaken to investigate the mechanism of liberation of tumor cells into the blood stream in connection with the process of tumor growth, using three strains of ascites tumor such as Yoshida sarcoma (YS) with infiltrative growth pattern, AH100B with expansive one, and AH109A with intermediate one. For this experiment, a new method for the quantitation of the number of circulating tumor cells was devised. From its results, it was concluded as follows: (1) Tumor cells appear first in the circulating blood at the transitional phase from logarithmic growth to declining growth. (2) Tumor size is a macroscopical index of the risk of the liberation of tumor cells into the blood stream. (3) Tumor necrosis is a histological sign suggesting that the tumor growth phase is declining, namely, that hematogenous dissemination of tumor cells has begun already. (4) There is a striking difference in the frequency and number of tumor cells in the venous blood among these three tumor strains. The tumor strains in order of the number of the circulating tumor cells are YS, AH109A, and AH100B. (5) In YS and AH109A, the transition of the number of tumor cells liberated into the blood stream is closely related to the growth process of tumor tissue. (6) Liberated cells of AH109A and AH100B disappear promptly from the blood stream.

View full abstract

The effect of 60-F, a fraction obtained by 0.5∼0.6 saturation of ammonium sulfate of streptomycin-pretreated cell-free extract from live hemolytic streptococci (avirulent Su strain), on release of ⁵¹Cr from the ⁵¹Cr-labeled Ehrlich ascites carcinoma cells was studied with following results:
a) 60-F was found to be highly effective in releasing ⁵¹Cr from ⁵¹Cr-labeled Ehrlich ascites carcinoma cells.
b) Additionally, destructive pictures of the tumor cells contacted with 60-F in vitro was observed by phase-contrast microscopic examination.
c) Indication was that the ⁵¹Cr-releasing assay method is also useful for the study of the direct cytolytic effect of 60-F.

View full abstract

B6C3F₁ female mice fed 100 or 200ppm of the aromatic amine, toluene-2, 4-diamine, developed significant number of carcinomas of the liver. The carcinomas varied from well-differentiated to poorly differentiated. Treated mice also developed hyperplastic hepatic nodules, but cirrhosis was not observed. Hyperplastic nodules, carcinomas, and cirrhosis of the liver have been described in rats ingesting toluene-2, 4-diamine.

View full abstract

Transplantation of fetal mammary gland mesenchyme into mammary glands of 2-month-old syngeneic virgin mice resulted in focal re-enactment of events that normally occur probably during fetal and early postnatal development of the mammary gland. Portions of the recipient's mammary duct system in contact with the fetal mammary mesenchyme underwent branching and proliferation in a pattern resembling that of rudimentary mammary gland development. This process occurred in C3H mice regardless of whether or not the milk-transmitted mammary tumor virus (MTV-S) was present. In mice carrying MTV-S, mammary cancers of Types A and B appeared earlier and more frequently in the mammary glands that had received transplants of fetal mammary mesenchyme, compared with those in the glands that received no fetal mesenchyme. Some of the smaller cancers were shown to develop directly from portions of the mammary gland interacting with fetal mammary mesenchyme, without preformation of typical hyperplastic alveolar nodules. In C3H mice not carrying MTV-S, cancers did not appear in the similarly treated mammary glands. These facts suggest that non-hormonal and probably nonviral factors that stimulate focal proliferation in the mammary duct system resulting from transplantation of fetal mesenchymes eventually accelerate local development of mammary cancers.

View full abstract

An experimental study was made on the suppressive effect on lymph node metastasis of an antitumor agent administered during the clamping of the portal vein. When mitomycin-C was injected into the tail vein of the rat and the portal vein was simultaneously clamped, a higher concentration of the drug was detected in the mesenteric lymph nodes compared to the conventional intravenous administration. The growth of mesenteric lymph node metastasis was markedly suppressed by the combined use of mitomycin-C and portal vein clamping. These results suggest that the procedure applied in the present study directs a high concentration of antitumor preparations to the lymph nodes in the portal vein region, and thus is a good method for the suppression of lymph node metastasis.

View full abstract

In 36 patients with advanced breast cancer, most of whom had been subjected to major endocrine ablation therapy such as bilateral adrenalectomy, correlation between the presence or absence of estrogen receptor (ER) in tumors and response to chemotherapy was investigated. Complete or partial response was obtained in 6 of 14 (42.6%) patients with ER-positive tumors and in 3 of 22 (13.6%) patients with ER-negative tumors (P=0.1). The response to chemotherapy was not influenced by the concentration of ER in tumors. Distribution of the duration of regression readings and the survival time was not significantly different between the patients with ER-positive and -negative tumors. Some regimens which did not include adriamycin, such as MFC (mitomycin-C, 5-fluorouracil, and cytosine arabinoside) had a good effect on the ER-positive cases, whereas the response to regimens including adriamycin was not influenced by the ER positiveness of the tumors. In the classification of some clinical parameters of the patients, response of the patients with ER-negative tumors does not seem to be better than that of the patients with ER-positive tumors. Responses to the major endocrine ablation therapy and also to the subsequently carried out chemotherapy were influenced by the presence or absence of ER in tumors before the endocrine therapy.

View full abstract

In vitro and in vivo transformed epithelial cells of the rat urinary bladder keratinized well on a coverslip culture. Keratinization proceeded more rapidly in in vitro-transformed cells than in in vivo-induced bladder cancer cells. The grade of keratinization was estimated from the absorption spectrum of a Papanicolaou-stained specimen which afforded two peaks derived from keratinized cells and viable cells. Using this semiquantitative method, effect of various drugs on keratinization was studied on 3 lines of in vitro-transformed epithelial cells. Vitamin A and its analogs prevented the keratinization in 2 out of 3 lines at dosage over 1μg/ml, but other drugs such as vitamin C, E, and K, steroid hormones, cyclic AMP, polyamines, polyanions, and dimethyl sulfoxide were ineffective for preventing keratinization. Amino acid compositions of keratinized cells and viable cells were not fundamentally different.

View full abstract

A temperature-sensitive strain of Sendai virus, HVJ-pi, showed little or no cytopathic effect and led to establishment of carrier cultures in several cell lines. By the use of this characteristic, L1210 leukemia cells persistently infected with HVJ-pi (L1210/c-HVJ-pi) was established, almost all of which were positively stained with fluorescent HVJ antibody. They are viable and grow almost equally as uninfected L1210 leukemia cells in vitro. Athymic nude mice (BALB/c, nu/nu), deficient of T-cells, died from intraperitoneal inoculation of L1210/c-HVJ-pi cells as well as by uninfected L1210 leukemia cells. However, viable L1210/c-HVJ-pi cells showed lower transplantability in normal syngeneic mice. This immunological mechanism of rejection was explained by the modification of cell surface membrane due to HVJ-pi infection. The mice which survived the inoculation of 10⁵ L1210/c-HVJ-pi cells were able to reject 10⁵ uninfected L1210 leukemia cells challenged subsequently. The induction of immune resistance was more prominent in (C57BL/6×DBA/2) F₁ mice or (BALB/c×DBA/2) F₁ mice than in DBA/2 mice.

View full abstract

Another Kasahara-variant alkaline phosphatase isoenzyme was found in 2 out of 25 human renal cell carcinoma tissues. This enzyme electrophoresed in a single diffuse band which is cathodal to but continuous with the liver alkaline phosphatase. After neuraminidase treatment, this enzyme electrophoresed in the same position as that of neuraminidase-treated Kasahara isoenzyme. The enzymic properties of another neuraminidase-treated Kasahara-variant enzyme such as inhibitions by L-phenylalanine, L-homoarginine, L-tryptophan, and L-leucine, effects of inorganic phosphate, urea, and sodium dodecyl sulfate, heat stability, and the reactivity with concanavalin-A are consistent with those of Kasahara isoenzyme. On Ouchterlony's double diffusion, the precipitin lines of Kasahara and the new variant enzyme produced by antibody to Kasahara isoenzyme fused completely. These facts may indicate the occurrence of another Kasaharavariant isoenzyme.

View full abstract

Intestinal metaplasia in human stomach was distinguished macroscopically into sucrase-positive and trehalase-positive areas, and sucrase-positive and trehalase-negative areas, by location of these disaccharidase activities with TES-Tape. After location of these two areas with TES-Tape, tissues were taken from them for colorimetric measurement of sucrase, trehalase, leucine aminopeptidase (LAP), and alkaline phosphatase (ALP). Results showed that in the mucosa from sucrase-positive and trehalase-negative areas, trehalase activity was not detectable and the activities of sucrase, LAP, and ALP were lower than in sucrase-positive and trehalase-positive areas.

View full abstract

Intraperitoneal inoculation of either levamisole or 2×10⁶ cells of concanavalin-A (Con-A)-bound L1210 leukemia vaccine produced no cured mice after subsequent ip inoculation of 10³ live L1210 cells. Combined inoculation of levamisole and Con-A-bound vaccine produced about 20% cure incidence but no prolongation of life span of tumor-bearing mice after inoculation of live L1210 cells. Combined inoculation of levamisole and Con A-free vaccine did not induce detectable immune resistance in mice. These results suggest that levamisole enhanced host response to cell-bound Con-A associated with immunogenic potency of the vaccine. Levamisole given intravenously and orally was as effective as that inoculated intraperitoneally in enhancing the induction of resistance in mice by Con-A-bound vaccine. Dose of levamisole was very critical for this enhancement, being effective at 0.38mg/kg but not either at 0.75 or 0.19mg/kg. Furthermore, levamisole restored immune resistance induced by a larger inoculum of Con-A-bound vaccine cells (10⁷) and impaired by cyclophosphamide.

View full abstract

This experiment was carried out to see the effect of aging on the induction of gastric carcinoma in rats by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Three groups of rats of 3 different ages, 6, 20, and 40 weeks old, were used. Comparison of incidence of gastric carcinoma in these groups revealed a significant difference. Tumor incidence of the gastrointestinal tract of rats were 94.7, 73.8, and 48.8%, respectively, at 6, 20, and 40 weeks of age. The incidence of adenocarcinomas of glandular stomach was also significantly decreased with the advance in age.
However, there was a significant difference in the intake rate of MNNG per gram body weight between the young (6 weeks of age) and old rats for the first 25 weeks during the experiment which may account for the difference in tumor incidence between young and old rats. However, even when the young rats were administered a decreased amount of MNNG, same as that in old rats, they also showed higher incidence.
From these results, it was concluded that the amount of MNNG ingested was not responsible for the decreased incidence of gastric adenocarcinoma in older rats. Aging might be the primarily the cause of low incidence.

View full abstract

HeLa cells contain two forms of DNA polymerase-α (P-I and P-II) with varied affinity to DNA, separable on a DNA-cellulose column. The activity of each form was followed during a cell cycle of synchronized culture of the HeLa cells. P-I was recovered from the nuclear extract and P-II from the cytoplasmic fraction. The P-I activity remained at a low level during M to Gl phase until a marked increase between late Gl and S phases, while P-II activity increased gradually throughout the period. Both activities attained their highest level at mid-S-phase and then the P-I activity declined more rapidly than the P-II. Addition of hydroxyurea at mid-S phase inhibited the decrease of both enzyme activities.
The enzyme activity of nuclear extract from S phase cells was not inhibited by mixing with extract from M phase cells. When the cytoplasmic fraction from M phase cells was chromatographed on a DNA-cellulose column, single activity peak was observed at the position of P-II. These results suggest that the decrease in P-I activity is neither due to the presence of an inhibitor nor to mere release of the enzyme from chromosomes.

View full abstract

Cytostatic activity of peripheral blood monocytes against cultured cell lines of bronchogenic carcinoma was examined in patients with lung cancer. Cytostatic activity in lung cancer patients was neither augmented nor suppressed as compared with that of controls such as normal healthy persons, patients with malignancies other than lung cancer, and patients with benign respiratory diseases. There was no correlation between the cytostatic activity of monocytes and the advance of clinical stages of the disease. Conventional modalities of anticancer treatments such as surgery, radiotherapy, chemotherapy, and their combination therapy had no effect on cytostatic activity of peripheral blood monocytes. However, adequate immunotherapy with Nocardia rubra cell-wall skeleton augmented the cytostatic activity of peripheral blood monocytes, although adequate immunotherapy with Mycobacterium bovis BCG cell-wall skeleton had no effect.

View full abstract

Specificity of micro-leucocyte adherence inhibition (LAI) test was studied in patients with gastric cancer. Because of the presence of nonspecific reactions, specificity appeared obscure unless interaction analysis was applied to the results of LAI tests. Application of interaction analysis revealed that only leucocytes from patients with gastric cancer showed specificity to gastric cancer antigen.
Further technical improvement is required to eliminate nonspecific reactions which seem to be due to either leucocytes or tumor antigens.

View full abstract

Effect of Neocarzinostatin (NCS) on microtubules was examined both in vitro and in vivo. Although NCS had no effect on microtubule assembly in vitro nor dissociated cytoplasmic microtubular network in vivo, microtubular network appeared to be changed to coarse form in NCS-treated 3T3 and HeLa cells. Effect of NCS on microtubules was discussed in comparison with colchicine.

View full abstract

Studies were made on the maximal non-carcinogenic dose of dimethylnitrosamine (DMN) in rats. Groups of Wistar strain rats of both sexes, 6 weeks old, were given standard diet without DMN (group 1), or containing 0.1ppm DMN (group 2), 1.0ppm DMN (group 3), or 10ppm DMN (group 4) for 96 weeks and then sacrificed for hematological, serum-biochemical, and histopathological examinations.
After 96 weeks, the weights of the body and main organs in the different groups were not significantly different. The leucocyte count and blood urea-nitrogen (BUN) in group 4 were slightly increased, but other serum findings were not significantly different in different groups.
Hepatocellular carcinomas were found in group 3 (1 male and 3 females), but not in group 2. Hemangioendotheliomas of the liver, adrenal adenomas, pituitary adenomas, interstitial cell tumors of the testis, ovarian tumors, and leukemia were also found. Pyelonephritis was found in both experimental and control animals, but no kidney tumors developed with these dose levels of DMN.
These results show that on long-term oral administration to rats, 1.0ppm DMN is the minimum carcinogenic dose, while a level of about 0.1ppm DMN is non-carcinogenic.

View full abstract

The movement of rat ascites tumor cells of AH66F and Yoshida sarcoma cultured in an agar medium was analyzed. Contraction occurred at the frontal fringe of each cell, and the cytoplasm and nucleus seemed to pass through the site of contraction. Morphological changes observed were similar to that of the leucocyte.

View full abstract

Electron microscopy of thin sections of syncytia, formed by fusion of homologous XC cells, clarified that these heterologous cells were simply invaginated by syncytia and that they were clearly separated by the limiting cytoplasmic membranes. The C particles found at the boundary showed no sign of connection with the surrounding syncytial cell membrane, while some were seen budding from the surface of the producer cells. These results strongly suggest that the XC cells, even when cocultured with the C-type virus-producing myeloma cells, fuse homologously among themselves, and the virus-producing heterologous cells do not participate in the event.

View full abstract

A new cell line was established from a transplantable rat stomach cancer induced by Nmethyl-N'-nitro-N-nitrosoguanidine, which might be profitable for a single cell cloning to analyze the multidirectional differentiation of rat stomach cancer cells and also a useful in vitromodel for the comparative study of human stomach cancer.

View full abstract

Level of ³H-dexamethasone binding to cytosol proteins in the regenerating liver decreased singnificantly 24hr after 70% hepatectomy when rapid DNA synthesis occurs. Increased binding was observed after 5 day in adrenalectomized and 70% hepatectomized rats, but enhancement of the binding was not prominent in rats with intact adrenals.

View full abstract