An intron fragment of a rat glutathione S-transferase P (GST-P) genomic clone was used to assign the chromosomal localization of the GST-P gene. In situ hybridization analysis with the genomic DNA indicated that the GST-P gene was localized on band q43 of rat chromosome 1.

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A novel v-yes-related proto-oncogene, syn, was assigned to region q21 of human chromosome 6 by in situ molecular hybridization. The present regional mapping substantiated the previous assignment that was performed by analyses of human-mouse somatic cell hybrids and filter blot hybridization.

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N-myc is a member of the myc oncogene family and has been thought to be specific to neurogenic cells, since it is amplified in some neuroblastomas, retinoblastomas and small-cell lung carcinomas with endocrine properties. Hybridization analyses of DNAs isolated from surgically removed tumors revealed that N-myc was amplified about 6-fold in one of the three embryonic rhabdomyosarcomas examined. The rhabdomyosarcoma containing the amplified N-myc had metastasized into bone marrow, which is preferentially involved in the metastasis of neuroblastomas but is usually not involved in the case of rhabdomyosarcomas. Since rhabdomyoblasts are derived from mesenchyme, this indicates that N-myc gene amplification is not restricted to neurogenic tumors.

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Rats of the BUF/Mna strain developed spontaneous epithelial thymomas morphologically indistinguishable from human homologues at virtually 100% incidence. Segregation of thymoma development among crosses between BUF/Mna and ACI/NMs, which has 0% thymoma incidence, indicated that thymoma susceptibility was determined principally by a single autosomal dominant gene Tbm-1 (thymoma in BUF/Mna rats). In these crosses, another autosomal dominant or semidominant gene(s) contributed by ACI/NMs parents moderately reduced the thymorna incidence.

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The arrangements of the T cell receptor (TCR) β genes were studied in leukemias with immature T cell phenotype. Three cases of acute lymphocytic leukemia (ALL) and one case of chronic myelocytic leukemia in blastic crisis (CML-BC), which expressed only Tp40 antigen of cluster of differentiation (CD) 7 without erythrocyte rosette receptor (E), did not show rearrangements of TCR β chain genes. Two of 5 ALL cases which expressed an additional T cell antigen, TI of CD5, showed rearrangements of TCR β genes. Two cases of CML-BC expressing T1 and Tp40, however, had unrearranged TCR β genes. The results altogether showed that a part of E^- T1⁺ Tp40⁺ ALL cases is of T cell origin.

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Urine of rats treated with promoters of urinary bladder carcinogenesis was analyzed during weeks 8 to 24 of administration. The sodium salts of several chemicals, including ascorbic acid, erythorbic acid, acid saccharin and o-phenylphenol increased the urinary pH and sodium ion concentration of the urine. In contrast, treatment with sodium hippurate did not cause elevation of urinary pH although it increased the sodium ion concentration in the urine. Butylated hydroxyanisole, butylated hydroxytoluene (BHT), and ethoxyquin did not affect the urinary pH or any electrolytes except for an increase of phosphorus in the urine of rats given BHT or ethoxyquin. Scanning electron microscopic examination showed that epithelial cells of the urinary bladder of rats given promoters of urinary bladder carcinogenesis had pleomorphic microvilli, short, uniform microvilli, and ropy or leafy microridges on their surfaces. Thus, for the class of promoters including the sodium salts of weak to moderate acids, the elevation of urinary pH and the increase of sodium ion concentration accompany the promoting activity, whereas these changes do not occur following administration of the antioxidant class of bladder tumor promoters.

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Butylated hydroxyanisole (BHA) was given to F344 rats, Syrian golden hamsters and B6C3F₁ mice at 2 doses for up to 104 weeks. The two doses were 2.0% and 1.0% for rats and hamsters, and 1.0% and 0.5% for mice. Animals were sacrificed sequentially at 8-week intervals from week 8 to week 104, and the carcinogenic effects of BHA on the forestomach were examined histopathologically. Papillomas and carcinomas were found in rats, hamsters and mice. In rats, papillomas first appeared in week 8 in the group given the higher level of BHA and in week 56 in that given the lower level. The first carcinoma was observed in week 48 in rats given the high level, while no carcinoma was observed in rats given the lower level. In hamsters, papillomas appeared in week 8 in both BHA-treated groups, and in both groups, the incidence of papillomas was much higher than in BHA-treated rats. Squamous cell carcinomas were observed in 4 hamsters (10.0%) among those that survived more than 64 weeks on treatment with the higher level of BHA and in 4 (7.3%) among those treated with the lower level. In mice, papillomas were induced by BHA in both BHA-treated groups after more than 88 weeks. Although the incidence was not statistically significant, carcinoma was also seen in mice, suggesting that BHA may also be carcinogenic to mouse forestomach.

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The effects of a single intragastric application of dibutyltin dichloride (DT), at a dose of 30mg/kg body weight, on N-nitrosobis(2-oxopropyl)amine (BOP)-induced pancreatic carcinogenesis were studied in female Syrian golden hamsters. DT, which has been shown to selectively induce bile duct injury, was administered either 1 week before or after BOP initiation. BOP was injected subcutaneously once a week for 5 weeks at a dose of 10mg/kg body weight. Controls were injected with BOP alone or given DT without carcinogen. Animals sacrificed at the end of the 25-week experimental period showed a significant inhibitory effect of DT on pancreatic carcinoma induction when DT was given before BOP treatment, although no such influence was evident with DT treatment following BOP exposure. These results indicate that the bile duct and more especially common bile-duct injury induced by DT may be relevant to the inhibition of the initiation stage of BOP-induced pancreatic carcinoma development in Syrian hamsters.

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The histopathological effects of hyperthermia with or without radiation were investigated in a transplanted mouse mammary carcinoma. Since the histopathological changes following hyperthermia differed greatly between the center and periphery of a tumor, we analyzed the changes in each area separately according to a semi-quantitative method developed by us. Three days after hyperthermia at 44°for 45min, undamaged tumor cells were found mostly in the tumor periphery adjacent to normal tissues. This phenomenon was observed when the entire tumor could be heated almost homogeneously. When a tumor was treated by heat alone, the thermal damage disappeared 7 days after treatment. On the other hand, treatment with hyperthermia plus radiation caused pronounced damage in the tumor center and tumor periphery 7 and 14 days after treatment. These combination effects depended on the radiation dose. The present findings demonstrate that combination of high doses of irradiation and hyperthermia is very effective for potentiating the thermal damage in the tumor periphery.

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The effects of step-up (42→44°sequence) and step-down (44→42°sequence) heating were studied on a transplantable mammary adenocarcinorna of C3H/He mouse. Tumor-bearing legs were immersed in a water bath and the response to hyperthermia was evaluated in terms of the delay in tumor growth. Tumor growth was delayed greatly with increase in the duration of treatment with 44°hyperthermia, whereas with 42°hyperthermia of up to 180min, tumor growth was delayed only slightly. The effects of step-up heating were similar to those of 44°hyperthermia alone and the response was enhanced by a factor of 0.9-1.1 with the 60-min treatment at 42°followed by treatment at 44°. Thermal resistance developed when the preheating time at 42°was longer than 30min. On the other hand, the tumor response was markedly enhanced by step-down heating by a factor of 1.8-2.4 with the treatment at 44°followed by 60-min treatment at 42°. Since the enhancement factor for skin damage found previously was similar to that for the tumor, therapeutic gain cannot be expected by the use of these combined heat treatments.

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Evidence has been accumulated that the phorbol ester receptor in intact cells is protein kinase C. However, it is not certain whether the various actions of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured cells are all mediated by the activation of protein kinase C. Therefore, we examined the effects of inhibitors of protein kinase C, palmitoylcarnitine (PC) and phloretin (PH), on several actions of TPA on cells. PC at the concentration of 30μg/ml completely prevented the inhibitory actions of TPA on differentiation of Friend leukemic cells (FLC) induced by hexamethylene bisacetamide (HMBA) and on metabolic cooperation of V79 cells. PC, however, did not prevent the TPA-induced promotion of the transformation of BALB/3T3 cells, even at the concentration of 40μg/ml. On the other hand, PH at the concentration of 30μg/ml did not inhibit the actions of TPA to inhibit differentiation of FLC and metabolic cooperation of V79 cells, but completely inhibited the transformation of BALB/3T3 cells and its promotion by TPA. These results indicate that protein kinase C possibly mediates some of the actions of TPA, such as the inhibition of differentiation and metaboliccooperation, but not that of the promotion of in vitro transformation.

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A monoclonal antibody (GC 302) established in our laboratory, which was reactive with gastric carcinoma and other epithelial carcinoma but not with normal gastric mucosa or other malignant tumors of mesenchymal origin, was used to investigate the radioimmunolocalization of tumors. Various kinds of target cells (5×10⁵) were incubated with ¹²⁵I-labeled GC 302, and radioactivity was determined with a gamma counter. It was shown that there was a 500-to 1, 000-fold increase in counts for gastric carcinoma (NUGC-2, NUGC-4, MKN-28 and MKN-45) as compared to those of normal lymphocytes and about 100-fold increase as compared to melanoma or leukemia. These findings were consistent with those obtained from the study of immunohistochemistry using GC 302. An in vitro assay was also carried out using nude mice bearing gastric cancer and inoculated with ¹²⁵I-labeled GC 302. There was a 2- to 3- fold increase in radioactivity in the tumor and a 4-to 5-fold increase as compared with the visceral organs. Although the tumor: blood ratio was relatively low, radioimmunoscintigraphy could be done successfully with the aid of computed radiography. We thus conclude that further testing of GC 302 is worthwhile to establish whether or not it is useful for radioimmunoscintigraphy of metastatic lesions of gastric cancer for possible clinical application.

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Production of human monoclonal antibodies reactive to stomach cancer was attempted by the hybridoma technique using splenic lymphocytes from stomach cancer patients. The parental cells used were NS-1 mouse myeloma line and three human lines including RPMI-1788 6TG^R, which was established in our laboratories. Ten mouse-human and two human-human (from the fusion with RPMI-1788 6TG^R) hybridomas have been producing IgM antibody for over 18 months, and all the heterohybridomas yielded ascites when transplanted into nude mice. Four antibodies produced by the heterohybridomas were selected and analyzed. These 4 antibodies, 3F6, 4A10, 3H5 and 1F9, reacted predominantly to cytoplasmic antigens of stomach and other epithelial cancer lines. The reactivity against human tumors transplantable in nude mice showed that all antibodies but 3F6 were reactive with stomach and lung cancers. Smears prepared from normal and cancer tissues were also tested, and these 4 antibodies showed positive reactions not only to stomach cancer, but also to normal stomach and colon. The reactivity against fetal tissues demonstrated that 3H5 antibody was reactive with epithelium of the stomach, and 1F9 antibody was positive with epithelium of the respiratory tract and bile duct, but the other two were negative. Thus, the serological analysis showed that the antigens detected are not tumorspecific, but are differentiation antigens. Chromosome analysis of these 4 mouse-human hybridomas and another one, which seems to produce an antibody against keratin, showed that three retained human chromosome 14 on which immunoglobulin heavy chain (Ig H) gene is located, but two did not. Southern blot analysis, however, revealed that all 5 hybridomas had a human Ig H gene.

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A large number of fibrosarcoma cell lines was established in vitro from a tumor mass induced freshly by inoculating 3-methylcholanthrene (MCA) subcutaneously (sc) into C3H/HeN mice, and more than five clones were isolated from each cell line by the limiting dilution technique. The present study investigated a) qualitative and quantitative comparison of the immunogenicity [tumor-associated transplantation antigen (TATA) activity] of different tumor clones and b) the relationship between such immunogenicity and the expression of H-2 class I antigens. When TATA were compared between different clones from the same tumor, these TATA were revealed to be cross-reactive to each other. On the other hand, the comparison of TATA between clones from different tumors demonstrated the existence of individually unique TATA in these clones. In addition to qualitative heterogeneity of TATA from different tumors, the magnitude of immunogenicity was also heterogenous in the individual clones established. Whether or not such quantitative heterogeneity of immunogenic strength was related to the expression of H-2 (class I) antigens was examined by flow microfluorometry studies using anti-H-2^k antibodies. The results demonstrated that there was no correlation between TATA activity capable of inducing in vivo tumor resistance and the expression of H-2 antigens. This contrasted with parallelism between the expression of H-2 antigens and inducibility of cytotoxic T lymphocytes (CTL) or lysability of tumor cell clones by CTL. These results are discussed in the context of the cellular mechanism of tumor cell eradication in vivo and the regulation of cell surface H-2 expression in vitro and in vivo.

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The mechanism by which tumor-specific Lyt-1⁺2^- T cells exhibit their antitumor effect in collaboration with an adherent cell population was investigated with the use of a double diffusion chamber. The double diffusion chamber was prepared by separating the two chambers from each other by a Millipore membrane and implanted in the peritoneal cavity of C3H/He mice. When one chamber contained normal C3H/He spleen cells plus syngeneic viable MH134 hepatoma cells and the other contained normal C3H/He spleen cells plus syngeneic viable X5563 plasmacytoma cells, tumor cells in both chambers continued to proliferate. In contrast, the injection of spleen cells immunized to the MH134 tumor into one (the first) chamber containing MH134 tumor cells not only resulted in the growth inhibition of MH134 tumor cells, but also exhibited an appreciable inhibitory effect on the growth of X5563 tumor cells admixed with normal spleen cells in the other (second) chamber. The growth inhibition of X5563 tumor cells in the second chamber was mediated by Lyt-1⁺2^- T cells specific for MH134 tumor cells admixed in the first chamber. Such tumorspecific Lyt-1⁺2^- T cell function was dependent on the existence of adherent cells in the first chamber, and adherent cells in the second chamber were also required for the X5563 growth inhibition. In addition, when the second chamber containing adherent cells, instead of the connection to the first chamber, was provided with macrophage-activating factor (MAF), X5563 growth inhibition was also observed. These results indicate that adherent cells are required for activating tumor-specific Lyt-1⁺2^- T cells and for functioning as nonspecific antitumor effector cells activated by factor(s) such as MAF from Lyt-1⁺2^- T cells.

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The growth-inhibitory activity of human recombinant γ-interferon (rIFN-γ: GI-3) against 30 human cultured cell lines derived from leukemias and lymphoma (9 T-cell, 13 B-cell, 2 nonTnonB and 6 myeloid cell lines) was measured quantitatively by in vitro regrowth assay. Only three myelomonocytoid cell lines (HL-60, U937 and THP-1-0) were found to be sensitive, and HL-60 was the most sensitive. However, the sensitivity of HL-60 was found to be much influenced by both the batch and the concentration of fetal calf serum (FCS) used in the experiment. In the experiments using a certain FCS, GI-3 had a high antiproliferative activity against HL-60 at the optimal concentration (10⁴-10⁵ U/ml), when assayed in medium containing 10% FCS. Both lower and higher concentrations of the interferon than 10⁴-10⁵ U/ml resulted in decreased antiproliferative activity. When a high concentration (30%) of the FCS was employed in the assay, the antiproliferative activity of GI-3 was also much reduced. In the experiments using the other FCS, no antiproliferative activity of GI-3 against HL-60 was observed on assay in the medium containing 10% FCS, but significant antiproliferative activity was observed in the medium containing 30% FCS from the same source. Experiments using serum-free culture medium revealed that GI-3 itself had both a slight growth-inhibitory action at the optimal concentrations (at about 10⁴ U/ml) and growth-enhancing activity at high (10⁶U/ml) concentration. The antiviral titer of GI-3 was stable during 72hr of incubation in growth medium with or without cells. These findings suggest that there are cofactors in the serum which positively or negatively influence the growth-regulatory activity of GI-3 against HL-60 cells.

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Monoclonal antibody against tumor-associated antigen (TAA) expressed on bovine leukemia cells was conjugated to liposomes containing adriamycin (ADM), and the specificity and therapeutic effects of the conjugates were examined in vitro and in vivo using a TAA-positive bovine leukemia cell line as the target tumor. In vitro studies with the TAA-positive cell line clearly indicated that the antibody-conjugated liposomes containing ADM exerted selective effects on TAA-positive cells in the inhibition assay of ³H-thymidine incorporation. Three injections of liposomes containing ADM (4mg/kg) into tumor-bearing nude mice significantly inhibited the tumor growth and the therapeutic effect of the antibody-conjugated liposomes was far greater than that of normal mouse IgG-conjugated liposomes as assessed in terms of tumor size.

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