Co-cultivation of rabbit lymphocytes with a human cell line carrying adult T-cell leukemia (ATL) virus (ATLV) resulted in the establishment of an ATLV-producer rabbit cell line. This cell line expressed ATL-associated antigens and reacted with antisera to the structural proteins of human T-cell leukemia virus which is identical or closely related to ATLV.

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Incubation of 6-nitrobenzo [a]pyrene[7, 10-¹⁴C] and calf thymus DNA with rat liver S9 fractions resulted in covalent binding of nitrobenzo[a]pyrene to DNA. In the presence of flavin mononucleotide under anaerobic conditions, the amount of binding decreased. Binding studies using synthetic poly-nucleotides showed that there was a high preference for poly(dG) compared to poly(dA), poly (dC) and poly(T).

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Differences in the susceptibilities to sodium saccharin of the urinary bladder epithelium in various strains of rats and other animal species were examined. In Experiment 1, male ACI, Wistar, F344 and Sprague-Dawley rats were administered 5.0% sodium saccharin in the diet for 52 weeks. Rats were killed after weeks 12, 24, 36 and 52. In ACI rats, sodium saccharin induced not only preneoplastic lesions of the urinary bladder, but also tumors. However, in other strains it did not. The urinary bladder of ACI rats given sodium saccharin had the most marked lesions under scanning electron microscopy, with less marked changes in Wistar and F344 rats. Sprague-Dawley rats were resistant to sodium saccharin. In Experiment 2, male F344 rats, B6C3F₁ mice, Syrian golden hamsters, and Hartley guinea pigs were given 5.0% sodium saccharin in the diet for 20 weeks. The animals were sacrificed sequentially. Rats developed urinary bladder lesions during the experiment, as detected by light microscopy and scanning electron microscopy. Increased DNA synthesis of the urinary bladder epithelium was detected in rats by autoradiography. However, mice, hamsters and guinea pigs were resistant to sodium saccharin. These results indicate that there are strain and species differences in the urinary bladder response to sodium saccharin.

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Four groups of 21 male Wistar rats were given weekly subcutaneous injections of 1, 2-dimethylhydrazine at a dose of 20mg/kg body weight for 2, 4, 6 and 8 weeks, respectively, and 7 animals of each group were killed in weeks 24, 32 and 40. This treatment induced a clear dose-related increase in the total number of colonic carcinomas. The number of tubular adenocarcinomas, which were mostly located in the descending colon, was clearly dose-related, whereas the number of signet ring cell carcinomas, which developed in the ascending colon, was apparently dose-independent.

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The influence of sodium chloride on chemical carcinogenesis of the gastroduodenal tract was examined in male outbred Wistar rats exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the drinking water (100mg/liter) for 20 weeks. Sodium chloride given concomitantly with MNNG during the first 20 weeks of the experiment increased both the incidence and the size of tumors at 40 weeks. However, sodium chloride given after MNNG during the second 20 weeks of the experiment did not enhance tumor development. This study indicates that, although sodium chloride given with MNNG enhances tumor development, sodium chloride does not promote gastric carcinogenesis.

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Male F344 rats were continuously fed 0.05% phenobarbital (PB)- or 0.02% 2-acetylaminofluorene (2-AAF)-containing diet for 3 or 8 weeks. The hepatocytes were isolated by a collagenase-perfusion technique and changes in the phalloidin-sensitivity of the cells were examined. After an 8-week treatment with PB or 2-AAF, the phalloidin-sensitivity, in terms of formation of cytoplasmic blebs over the cell surface, was reduced significantly in both groups. The degree of insensitivity to phalloidin induced by PB was found to be similar to that induced by 2-AAF. Two weeks after cessation of the PB-feeding, the sensitivity had recovered to the control range, while the decreased sensitivity induced by 2-AAF persisted for at least 2 weeks after the cessation of 2-AAF-feeding. Furthermore, cytochemical examinations of normal and 2-AAF-treated hepatocytes revealed that the degree of sensitivity decreased in the order of normal hepatocytes, 2-AAF-treated γ-glutamyl transpeptidase-negative hepatocytes, and 2-AAF-treated γ-glutamyl transpeptidase-positive hepatocytes. The decreased sensitivity of the latter two cell types also persisted for 2 weeks after the cessation of carcinogen treatment.

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Oxidative in vitro metabolism of N, N-dibutylnitrosamine (DBN) by 9000g supernatant fraction (S9 fraction) prepared from rat liver and oxidation of DBN by chemical model systems (Udenfriend and Fenton) were investigated. The products retaining the N-nitroso group of DBN were analyzed by gas-liquid chromatography. Incubation of DBN with the S9 fraction gave hydroxy derivatives of DBN at the ω, ω-1, and ω-2 positions, while the chemical reaction of DBN in the Udenfriend and the Fenton model systems afforded compounds with an oxo group at the same positions. In all cases, ω-1 oxidation was predominant, followed by ω and then by ω-2 oxidations, while ω and ω-1 oxidations were the principal metabolic pathways of DBN in vivo in the rat. The differences in the oxidative transformation of DBN among in vitro enzymatic, non-enzymatic and in vivo systems are discussed.

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Enzymatic α-hydroxylation is believed to be the initial step in the metabolic activation of mutagenic and carcinogenic nitrosamines. Oxidative in vitro metabolism of N, N-dibutylnitrosamine (DBN) by hepatic microsomal fractions prepared from rats treated with phenobarbital and polychlorinated biphenyl (PCB) was investigated. Following incubation of DBN with the microsomal fractions, N-butyl-N-(3-hydroxybutyl)nitrosamine was identified by gas-liquid chromatography as the principal metabolite with the N-nitroso moiety, and butyraldehyde and/or two isomeric butyl alcohols (n- and sec-) were also identified and determined by gas-liquid chromatography and high-presssure liquid chromatography. The latter compounds originate from the unstable α-hydroxylated intermediate, N-butyl-N-(1-hydroxybutyl)nitrosamine, by spontaneous decomposition. An increased production of butyraldehyde with a concomitant enhancement of the mutagenicity of DBN toward Salmonella typhimurium TA 1535 was observed when microsomes prepared from phenobarbital- and PCB-treated rats were used. SKF 525-A not only inhibited the mutagenic activation of DBN by microsomes from rats treated with the inducers but also selectively inhibited the formation of butyraldehyde and butyl alcohols from DBN. These results indicate that among the four initial hydroxylations (α, ω-2, ω-1, and ω) of the butyl chain demonstrated in the metabolism of DBN, α-hydroxylation is primarily involved in the mutagenic activation.

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Species variations in response to urinary bladder carcinogens, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN), and N, N-dibutylnitrosamine (DBN), were investigated in several animal species from the metabolic point of view. Since N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN) and N-ethyl-N-(3-carboxypropyl)nitrosamine (ECPN) had been found to be the principal urinary metabolites which are responsible for the induction of bladder tumors by BBN or DBN and EHBN, respectively, in rats, acidic urinary metabolites with the N-nitroso moiety were isolated and determined by a colorimetric method after oral administration of these nitrosamines to rats, mice, hamsters, guinea pigs, and dogs. Qualitatively almost no species differences were observed among these animals in regard to the urinary metabolites except in the case of mice, in which the glycine conjugate of BCPN was isolated from the urine and identified as the principal metabolite of BBN and DBN. However, appreciable quantitative differences in the urinary excretion of BCPN or ECPN were found among these animal species, indicating that the differences in the susceptibilities of different animal species to urinary bladder carcinogenesis induced by BBN, DBN and EHBN may be closely related to the different extents of urinary excretion of the active metabolites of these nitrosamines.

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Mammary tumors have ceased to develop in descendants of one female of the RIII/AnOk mouse strain which was brought from the Mouse Colony of Okayama University Medical School in 1975, while descendants of another female of this strain have maintained a high tumor incidence. The former and latter descendants were separated out as RIII/AnOk/2 and RIII/AnOk/1 sublines, respectively. The amount of mouse mammary tumor virus (MMTV) in the milk of individual females in these two sublines was determined by means of a sensitive enzyme immunoassay. No MMTV antigens were detected in 59 milk samples of RIII/AnOk/2 females collected during the 1st to 7th lactation periods. On the other hand, all the milk samples of RIII/AnOk/1 females contained MMTV antigens with concentrations ranging from 9 to 400μg/ml. When RIII/AnOk/2 mice were foster-nursed by RIII/AnOk/1 mothers, the infected RIII/AnOk/2 females produced MMTV in the milk to almost the same degree as did the RIII/AnOk/1 females. No genetic differences between the two sublines were observed by the use of reciprocal skin grafting, tumor transplantation, and analysis of biochemical genetic markers. These results indicate that the arrest of mammary tumor development in the RIII/AnOk/2 subline was due to the disappearance of MMTV in the milk.

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A rat cell line, 3Y1, was transfected with the left-end DNA fragments of adenovirus (Ad) 31 DNA and transformed cell lines were established. A cell line 31BY4-1, which was induced by Ad31 BamHI-B (left-most 17.4% of the genome), showed typical transformation phenotypes, while cell lines 31GY1-2, 31GY2-5, and 31GY3-4, which were induced by Ad31 HindIII-G (left-most 6.7% of the genome), showed intermediate phenotypes between transformed and untransformed cells, with the following properties: (1) percent plating efficiencies in Eagle's minimum essential medium with 2% fetal calf serum and in soft-agar culture were extremely low, like those of 3Y1 cells; (2) they form tumors in newborn rats only after long latent periods. Southern blot hybridization revealed that, in all the transformed cell lines, viral DNA sequences were integrated at multiple loci in large-molecular-weight cell DNAs. Northern blot hybridization showed that viral mRNAs of early region 1A (ElA) and early region III (ElB) were both transcribed in 31BY4-1 cells as well as in KB cells early after infection with Ad31. On the other hand, ElB viral mRNA was present in a much lesser amount in 31GY cell lines, or was undetectable.

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Prostatic acid phosphatase (PAP) was purified from human seminal plasma by precipitation with ammonium sulfate and by serial chromatographies with concanavalin A-Sepharose, Sephadex G-100, and carboxymethyl-cellulose. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis, both with and without sodium dodecyl sulfate. The purified enzyme also gave a single precipitin line upon immunoelectrophoresis, when reacted with rabbit antiserum to seminal plasma. A simple method was developed for the preparation of monospecific antibody to PAP. In addition, a new immunoenzymatic assay system for PAP is reported, utilizing filter paper discs as a solid-phase to which monospecific antibody to PAP was covalently coupled. By this method, serum PAP in a range of 1 to 7, 000ng/ml could he accurately measured within 4hr.

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In order to study the biological nature of various mammary tumors, differences in the formation of polynuclear cells after the administration of cytochalasin B were investigated in cultures of human mammary tumors and normal mammary gland. When cytochalasin B was applied to the cultures, polynuclear cells increased in all cancer cases (6.1% on average), but relatively little effect was seen in cases of benign tumors and normal mammary gland (less than 1.1% on average). From these results, it appears that the difference in polynucleation on treatment with cytochalasin B may be useful as a biological means to distinguish human malignant and benign mammary tumors.

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A vitamin A analog, 13-cis-retinoic acid (13-cis-RA), was tested for effect on intercellular metabolic cooperation between 6-thioguanine-resistant and -sensitive V79 Chinese hamster cells. Most typical tumor promoters have been reported to inhibit metabolic cooperation. 13-cis-RA was found to enhance metabolic cooperation of cells treated with a potent tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate. However, inhibition of metabolic cooperation was noticed when 13-cis-RA was used at higher concentrations.

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A3/Kawakami was derived from ascitic lymphoma cells of a 68-year-old female patient with malignant lymphoma (non-Hodgkin's lymphoma, diffuse, large cell type) of the stomach and A4/Fukuda was derived from ascitic lymphoma cells of a 52-year-old female patient with double cancer of the colon (well-differentiated papillary adenocarcinoma and non-Hodgkin's lymphoma, diffuse, large cell type). The fresh ascitic lymphoma cells in the case of A3/Kawakami were surface immunoglobulin-positive, but the cultured A3/Kawakami cells no longer expressed any distinct markers. In the case of A4/Fukuda, the fresh ascitic lymphoma cells and cultured cells did not express any specific surface markers. Only 20% of A4/Fukuda cells were reactive with OKI1. However, a small amount of IgM could be detected in the cell extract and concentrated culture supernate of A4/Fukuda. In addition, A4/Fukuda cells heterotransplanted into anti-thymocyte sera-treated newborn hamster or athymic nude mice with a BALB/c genetic background were found to have weak surface immunoglobulin and distinct cytoplasmic immunoglobulin (γ, μ). These data suggest that A4/Fukuda cells share the characteristics of the late differentiation stage of B-cell lineage (intermediate between mature B-cells and plasma cells). It was found that monoclonal antibodies, OKT4 and anti-Leu 3a, which are known to react specifically with inducer/helper T-cells, reacted to both A3/Kawakami and A4/Fukuda cells. The karyotypes of both A3/Kawakami and A4/Fukuda cells were very complicated but included some marker chromosomes such as 14q+.

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Surgically obtained portions of the human gastrointestinal tract were perfused with oxygenated perfluorochemical blood substitute, Fluosol-DA (FDA), containing tritiated thymidine. This procedure of ex vivo autoradiography was performed at the RI Center by perfusion of the isolated organ with FDA from a reservoir 190cm above the organ through a cannula inserted into the main artery. Microautoradiographic sections revealed that the distributions of labeled nuclei in the normal epithelium of the esophagus, stomach, jejunum, colon and rectum coincided well with those reported previously. However, in the normal colonic mucosa in cases of familial adenomatosis, no expansion or shift of the proliferative zone to the upper part of the mucosa was observed. No significant difference was observed between the labeled figures of normal colonic mucosa of cases of familial adenomatosis and ordinary colonic cancer. This is the first report of ex vivo autoradiography of the gastrointestinal tract of man for studies on cell kinetics.

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A new technique for indirect measurement of microvascular pressure in normal and tumor tissue was developed, by using a microneedle which occludes the microvessels without disturbing the microcirculation system. Thus, the microvascular pressure either in normal subcutis or in tumor tissue can be measured precisely at the single vessel level. There was a considerable drop of the pressure in the smaller arterioles (less than 40μm in diameter) in proportion to their diameters, while the pressure profile in venules was almost flat irrespective of their diameters. The mean pressure of venules 10-50μm in diameter was 12.0±5.2 cm H₂O, and some preliminary data obtained in pressure measurement of tumor vessels are also presented.

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Peritoneal macrophages obtained from male BALB/c mice intraperitoneally injected with Nocardia rubra cell-wall skeleton (N-CWS) showed abundant superoxide anion and hydrogen peroxide release in response to opsonized zymosan used as a trigger. These N-CWS-induced macrophages also showed marked cytolytic activity against syngeneic Meth A fibrosarcoma targets. To examine the possible role of superoxide anion and/or hydrogen peroxide in the lysis of Meth A cells, N-CWS-induced macrophages were assayed with Meth A targets in the presence of superoxide dismutase, catalase, or both. However, addition of these agents to the assays, in doses which abolished detectable superoxide anion and/or hydrogen peroxide release from the effector cells, had no inhibitory effects on the cytotoxicity of N-CWS-induced macrophages. In contrast, both a protease inhibitor and actinomycin D could inhibit the cytolysis of Meth A targets by N-CWS-induced macrophages. These data raise the possibility that protease(s) rather than oxygen intermediates might be involved in the cytotoxicity of N-CWS-induced macrophages against tumor cells.

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The in vivo and in vitro effects of Nocardia rubra cell wall skeleton (N-CWS) on natural killer (NK) activity of spleen and peritoneal lymphocytes of C57BL/6 mice were studied. The NK activity of spleen and peritoneal lymphocytes against YAC-1 lymphoma cells and cultured B-16 melanoma cells peaked at 3 days afte intraperitoneal or intravenous administration of N-CWS, and returned to the normal value 7 days later. The NK activity of speen lymphocytes was augmented by in vitro incubation with N-CWS. The appropriate concentration of N-CWS for the in vitro stimulation of NK activity in spleen lymphocytes was 2-5μg/ml.

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C57BL/6J mice with autochthonous 3-methylcholanthrene-induced tumors were given combined therapy with 2, 000rad of local irradiation and regional injections of cell-wall skeleton of Nocardia rubra (N-CWS). The greatest suppression of the tumor growth and the highest survival rate were observed in the combined treatment group as compared to the non-treated control, regional injections of N-CWS alone and irradiation alone groups. In addition, pulmonary metastasis was significantly suppressed by the combined therapy.

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Experiments were performed to explore the influence of local chemoimmunotherapy by intratumoral administration of mitomycin C (MMC) and/or BCG on the survival rate, lymph-node metastasis, growth pattern of rechallenge tumor, and host immune function, using a host-tumor system consisting of C3H/He mice and syngeneic tumor MH134. Combined intratumoral regimens of MMC plus BCG yielded a significantly higher survival rate than those achieved with BCG or MMC alone. Furthermore, the incidence of metastatic involvement of regional lymph nodes was also remarkably reduced in the combined regimen group. The group given the combined MMC-BCG regimen also showed a marked suppression of the growth of rechallenge tumor after surgical removal of the primary tumor and showed a greater induction of tumor-specific immunity than that seen in the group given intratumoral BCG injection alone. Both the delayed-type hypersensitivity, as measured by the footpad swelling assay, and the antibody response in spleen lymphocytes, as estimated by the plaque-forming cell assay, were found to be substantially depressed following a systemic injection of MMC, whereas the intratumoral administration of MMC had little or no effect on these parameters.

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Peritoneal macrophages from mice injected with glycogen proliferated in vitro in the presence of cell-free tumorous ascites. DNA synthesis of macrophages was also induced by ascitic fluid, but did not occur in the absence of the fluid. However, macrophage growth was inhibited at a higher concentration of ascitic fluid (>20%). The growth stimulating activity of this fluid was stable on heat treatment. The adherent cultured cells that proliferated were typical macrophages, as indicated by nonspecific esterase staining, pinocytosis and phagocytosis. These macrophages showed cytolytic activity against a murine tumor in the presence of wheat germ agglutinin. However, their cytotoxicity with antitumor antibody decreased during the culture period. These results indicate that tumorous ascitic fluid contains a macrophage growth factor(s) and that macrophages cultured with, and induced to proliferate by, ascitic fluid can kill tumor cells in cooperation with lectin but not antibody.

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Enhancement of specific transplantation resistance to a syngeneic tumor (KMT-17) was observed in WKA rats treated with the antileukemia drug busulfan (BU) (15 or 8mg/kg) 5 days after immunization with X-irradiated KMT-17 cells. Similar enhancement was also produced by treatment with mitomycin C (1mg/kg), but not cyclophosphamide (40 or 20mg/kg), adriamycin (3mg/kg), or BU (4mg/kg). The therapeutic effect of BU on transplanted KMT-17 tumor in WKA rats was relatively weak, but the therapeutic efficacy of BU as an immunomodulator appeared to be almost equal to that of PS-K, lentinan, or neurotropin. By means of the tumor-neutralizing assay using spleen cells, a stronger antitumor immune response was observed in BU-treated tumor-bearing rats than in the BU-untreated control group. An improvement of the therapeutic effect was obtained by combining neurotropin with BU, although its monotherapeutic effect was insufficient.

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The blood supply in she central portions of solid Yoshida sarcoma tumors is poor, but these portions contain viable cells capable of tumor growth when transplanted into new hosts. Attempts were made to increase penetration into these portions in rats by an injection of prostaglandin E₁ together with lissamine green dye (used as a marker of drug penetration). The lissamine green dye defined the regions which could not readily be reached by blood-borne substances. Next, mitomycin C in place of lissamine green was administered together with prostaglandin E₁. The growth of Yoshida sarcoma cells transplanted subcutaneously was suppressed significantly in the rats given mitomycin C with prostaglandin E₁ compared with that in the rats given mitomycin C alone. The effect was greater when prostaglandin E₁ was administered by an intraarterial route than by an intravenous route (P<0.01). Five patients with hepatic metastases from colorectal carcinoma were treated with a one-shot infusion of mitomycin C together with prostaglandin E₁. Two patients showed objective evidence of improvement.

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The cytotoxic effects of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-nitrosourea hydrochloride, thio-TEPA, 4'-dimethyl-epipodophyllotoxin-β-D-ethylidine glucoside, bleomycin, cis-platinum, mitomycin C, adriamycin and carbazilquinone on asynchronous cells of established human bladder carcinoma lines (KK-47, KW-103 and RT4) were investigated by the in vitro colony formation technique. The dose-survival curves were classified into 2 types: biphasic type (bleomycin) and exponential decrease type (the others). These types and the 50 and 90% growth inhibition doses of the drugs may offer a guide to chemotherapeutic design as well as to the selection of possibly effective agents for bladder cancer therapy.

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