The content of glutathione S-transferase placental form (GST-π) was immunohistochemically analyzed in 100 cases of human stomach carcinoma of various histological types and compared with the content in the stomach mucosa of the human fetus, infant and adult. High levels of GST-π content were demonstrated in all stomach carcinomas except for signet ring cell carcinomas. High levels were also present in surface mucous cells and glandular cells of the stomach from a fetus aged 18 weeks, whereas only the parietal cells of fundic glands in the adult stomach showed slight staining for GST-π. These results indicate that the phenotypic expression of GST-π in human stomach carcinoma is oncofetal in character.

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To study the carcinogenic action of N-methyl-N-nitrosourea (MNU) on the stomach, MNU in distilled water at a concentration of 400ppm was provided as drinking water to F344 male rats for 25 weeks (group I) or 15 weeks (group II). Twenty weeks following the cessation of the administration, invasive adenocarcinomas were found in the glandular stomach in 100% of 16 rats in group I and 38% of 21 rats in group II. Bone formation occurred within the stroma of carcinoma in 5 rats in group I. No neoplastic lesions developed in the esophagus, forestomach or duodenum of any rat. Thus, MNU in drinking water selectively induces glandular stomach carcinoma in high incidence in these rats.

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We have examined the activities of Epstein-Barr virus (EBV)-specific memory T-cells of carriers of adult T-cell leukemia (ATL) associated retrovirus (HTLV-I) and non-carriers in an ATL-endemic area, all of whom were EBV-seropositive, by assay of EBV-induced B-cell focus regression. The result showed that immune suppression, as represented by lowering of the regression, was present in 18 (29%) out of 63 healthy HTLV-I carriers, in contrast to none of 63 matched control persons (healthy non-carriers). This finding suggests that a suppression of cell-mediated immunity is induced by a persistent infection with ATL retrovirus.

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The amount of infectious virus released into the culture fluid from human immunodeficiency virus (HIV)-producing MOLT-4 cells (MOLT-4/HIV HTLV-III) was measured as a function of the culture conditions and cell density. After 3 days, the daily yield of infectious virus per cell in the fluid of non-growing cells cultured at high cell density was about 15 times higher than the accumulated yield from growing cells cultured at low cell density. SDS-PAGE analysis also showed a similar difference in the amounts of major gag protein p24.

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The effect of neurotropin on human immunodeficiency virus (HIV) production in a virus-producing MOLT-4 (MOLT-4/HTLV-III) cell line was studied. Culture of the cells for 4 days in the presence of 100μg/ml of neurotropin resulted in a 72% decrease in the number of infectious virus particles released into the culture fluid. A pulse-labeling experiment to analyze the synthesis of HIV proteins suggested that neurotropin may affect the budding process of the virus.

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In a DNA-mediated transfection assay using NIH3T3 cells, the DNAs from two out of twelve human hepatocellular carcinomas gave transformants. The transforming gene was identified as hst, which was originally found in DNAs from stomach cancers and a noncancerous portion of stomach mucosa, the putative product being a growth factor. Transcripts were not detected in the original HCC1 and HCC2 upon Northern blot analysis. The possible mechanisms involved in the hst gene activation are discussed.

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We have isolated a human genomic DNA (designated human frt) cross-hybridizing with the v-ros oncogene of UR2 sarcoma virus. Sequencing analysis of this fragment revealed that this sequence contains a 123-base-pair exon-like structure surrounded by consensus sequences of splice acceptor and donor sites. The deduced amino acid sequence of this stretch was highly homologous to a portion of a tyrosine kinase domain of src family. The human frt sequence was found to be conserved in a wide variety of vertebrates. By using a humanmouse hybridoma panel, human frt was located on chromosome 13, while human c-ros-1 was located on chromosome 6.

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Repeated topical application of 3-methylcholanthrene to the backs of BALB/c mice lowered the tissue levels of lathosterol and provitamin D₃, intermediates in one of the cholesterol biosynthetic pathways (the Δ⁷ pathway), though the activities of lathosterol 5-desaturase and provitamin D₃ 7-reductase were similar to those of control animals. These results seemed to indicate that carcinogen treatment exerted a depressive effect at some earlier step(s) than lathosterol synthesis. However, the content of cholesterol in mouse skin was not lowered in these animals, suggesting that another biosynthetic pathway might be activated. When diazacholesterol, which is known to inhibit the conversion of desmosterol to cholesterol, was administered together with the carcinogen, a marked accumulation of desmosterol was observed compared to animals given only diazacholesterol. Since desmosterol is an intermediate in the pathway in which the Δ²⁴ double bond is reduced at the final step (the Δ²⁴ pathway), this seemed to suggest that the Δ²⁴ pathway was activated by carcinogen treatment.

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The clonal origin of radiation-induced myeloid leukemia was studied in mice with cellular mosaicism for phosphoglycerate kinase (PGK). A single whole-body X-irradiation of 3 Gy resulted in development of 11 myeloid leukemias in 198 mosaic mice (5.6%). PGK of spleen cells from 9 myeloid leukemias assayed gave only a single band on electrophoresis. PGK of red blood cells (RBC) from one myeloid leukemia gave a single band identical to that of PGK of leukemic cells, while PGK of RBC from 4 other myeloid leukemias gave two bands. From these results, we concluded that radiation-induced myeloid leukemias in mice were of single cell origin, and that the origin of some myeloid leukemias might be a progenitor cell capable of differentiating into both erythroid and myeloid series.

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In order to clarify the natural transmission route of human T-cell leukemia virus type I (HTLV-I) from mother to child, we have followed two groups of children with ages of 1 to 3 years who were nourished either with HTLV-I-infected breast milk, or with non-infected milk from sero-positive, HTLV-I carrier mothers. Tests for the presence of antibody against HTLV-I revealed that 4 of 6 children in the former group developed HTLV-I infection, while only 1 of 14 children in the latter group became infected. The difference in HTLV-I infection rate for the children in the two groups was statistically significant (P<0.01 by chi-square). Furthermore, 2 of 4 elder siblings in the former group developed HTLV-I infection, whereas only one of 8 elder siblings in the latter group became infected. The overall rate of HTLV-I infection of breast-fed children born to HTLV-I-carrier mothers was 25% (8/32) by 3 years of age. Five of 6 mothers with HTLV-I-infected cells in the milk also possessed infected cells in their peripheral blood. Conversely 5 of 6 mothers without infected cells in the peripheral blood possessed no infected cells in their breast milk, suggesting that HTLV-I-infected cells in the peripheral blood can enter the breast milk. None of the 8 breast-fed children born to carrier mothers whose peripheral blood and breast milk-borne cells were negative, developed HTLV-I infection, suggesting that HTLV-I transmission from mother to child is dependent upon the number of HTLV-I-infected cells in carrier mothers.

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Three lines of transgenic mice carrying hepatitis B virus (HBV) DNA were produced. In a pSHB1BB transgenic mouse carrying one copy of BamHI fragment of HBV with a deletion of BglII-BglII fragment, no RNA transcription was observed. Transgenic mice that had integrated either one or three tandem copies of HBV DNA produced RNA in various tissues except liver. However, neither initiation nor termination of transcription occurred at the correct place. Hepatitis B surface antigen and hepatitis B e antigen were not detectable in these transgenic mice. HBV DNA was methylated at all CCGG sequences, which might have resulted in aberrant RNA production. In two out of five transgenic mice HBV DNA was integrated into the Y chromosome. Although the frequency is unusually high, the mechanism is not known yet.

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DNA samples from eighteen rat hepatocellular carcinomas, including those induced by oral administration of dibutylnitrosamine (DBN) with butylated hydroxytoluene (BHT), or DBN with butylated hydroxyanisole (BHA), have been tested for the presence of transforming activity by transfection assay with NIH3T3 cells. Of the eighteen samples, only one from a tumor induced by DBN and BHT gave transformants. The activated oncogene was identified as rat N-ras by Southern blot analysis.

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In order to examine the effect of alteration in methylation of the c-myc gene on hepato-carcinogenesis, the extent of methylation of the c-myc gene was examined in 24 tissues of hepatocellular carcinoma (HCC), 24 adjacent non-tumor liver tissues from the same patients and 16 control liver tissues by the use of restriction endonucleases. The following results were obtained. (1) The c-myc gene from HCC tissue tended to be hypomethylated in comparison with that in non-tumor liver tissue from the same patient. (2) The c-myc gene from non-tumor liver tissue was hypomethylated to various degrees in comparsion with that in control liver tissues. (3) The CCGG site in the third exon of the c-myc gene tended to be more extensively hypomethylated in HCC tissues than in non-tumor and control liver tissues. These results suggest that hypomethylation of the c-myc gene may occur to various degrees before the appearance of HCC, and may be associated with hepatocarcinogenesis. Moreover, the hypomethylation in the third exon of the c-myc gene is probably important for the development of HCC.

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The effect of β-cyclodextrin-benzaldehyde (CDBA) on pulmonary metastasis in C3H/He mice was examined. In experimental metastasis that was induced by iv injection of 1×10⁶ RCT(+) cells, the highest inhibition was observed in the mice that were treated daily with CDBA (5mg/day) for 1 week before tumor cell inoculation and further treated for 3 weeks after inoculation, when compared with those in other experimental groups that were given only pretreatment or posttreatment. The inhibitory effect was dose-dependent. In spontaneous metastasis that was induced by sc injection of 3×10⁶ RCT(+) cells, the inhibition of metastasis was also observed in the mice treated with CDBA (5mg/day) in the same manner as described above. However, the development of the primary tumor was not inhibited. CDBA-treated tumor-bearing mice showed almost as much NK activity as normal mice. Furthermore, although injection of 5-fluorouracil suppressed this activity to about 50% of that in normal mice, the combined treatment with CDBA could maintain the NK cell activity at the normal level. The results suggested that the inhibition of pulmonary metastasis might be induced by a combined effect of CDBA; that is, the direct inhibition of tumors and the maintenance of NK cell activity.

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The arrangements of the T cell receptor (TCR) β genes were studied in lymph node specimens with the histological characteristics of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD), AILD-like T cell lymphoma (AILD-T), T-zone lymphoma, or Lennert's lymphoma. Eight of 11 cases with AILD or AILD-T showed clonal rearrangements of TCR β genes: all three AILD cases showed clonal rearrangements of TCR β genes, while five of 8 AILD-T cases exhibited clonal rearrangement patterns. Malignant lymphoma evolved in one AILD case showed the same rearrangement band of TCR β genes as its primary AILD lesion. The rearrangement of TCR β genes was also observed in 2 out of 3 cases with T-zone lymphoma and 2 out of 2 cases with Lennert's lymphoma. None of the cases studied, except one AILD-T case, exhibited clonal rearrangements of immunoglobulin heavy chain genes. The results suggested that a significant proportion of AILD, AILD-T, T-zone lymphoma and Lennert's lymphoma cases are malignancies of peripheral T cell origin.

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Culture of hydrocortisone (HC)-resistant C57BL/6 mouse thymocytes with recombinant human interleukin 2 (IL 2) allowed the proliferation of the thymocytes and resulted in the generation of lymphokine-activated killer (LAK) cells cytotoxic to a variety of tumor cells. The cytotoxic activity of the LAK cells was greatly reduced by treatment with anti-Thy 1.2 or anti-Lyt 2.2 monoclonal antibody and complement but not with anti-asialoGM₁ antibody plus complement. Fractionation of IL 2-stimulated thymocytes by means of Percoll density gradient centrifugation revealed that both cytotoxic activity and binding capacity to target cells were greater in the cells with lower density and larger size than in the cells with higher density and smaller size. These IL 2-activated thymocytes expressed higher levels of both Thy 1, Lyt 2 and lymphokine-activated cell-associated (LAA) antigens than unstimulated thymocytes, as indicated by a flow cytometric analysis. The frequency of LAK precursor cells was found to be 7.5 times greater in the HC-resistant thymocyte population than in total thymocytes, as determined by means of a limiting dilution method. The LAK precursor cells in HC-resistant thymocytes appeared to be Lyt 1^- (or dull Lyt 1⁺), L3T4^-, Lyt 2^-, asialoGM₁^- T cells, because elimination of bright Lyt 1⁺, Lyt 2⁺ or L3T4⁺ T cells from HC-resistant thymocytes had no effect on the generation of LAK cells. These results indicate that LAK cells from mouse thymocytes are Lyt 2⁺ T cells which are inducible from HC-resistant Lyt 2^- thymocytes.

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The chorioallantoic membrane of chick embryo was used to examine the chemosensitivity of the murine tumors, B16-F1 melanoma, B16-F10 melanoma, Meth-A fibrosarcoma, and Ehrlich carcinoma. The tumors were grown on the membrane, and the effects of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride, cyclophosphamide, dacarbazine, 5-fluorouracil, methotrexate, adriamycin, mitomycin C, vincristine, and cisplatin on the growth of the tumors were tested by iv injection into a vein of the chorioallantoic membrane or injection into the yolk sac. The use of chick embryo limits the time for drug exposure to 3 or 4 days, but Ehrlich carcinoma and Meth-A fibrosarcoma needed longer for the test; the use of irradiation from a cobalt source overcame the problem by increasing the growth rate of the grafts of these two tumors. There appeared to be a good correlation between the effects of the drugs on B16 melanomas grown in the eggs and in the original animals. Many compounds, including pro-drugs such as cyclophosphamide and dacarbazine, could be assayed in this way.

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The synergistic effects of combined chemotherapy with adriamycin (ADR) and cyclophosphamide (CY) on L1210 tumors in mice were potentiated by use of a streptococcal preparation, OK-432, in a time- and dose-dependent way. Some mice were cured by treatment with the three agents, and resisted a later challenge by L1210 but not P388 leukemia cells. This immunity was blocked by administration of antimacrophage agents or CY. The effects of OK-432 were also studied with mice sensitized by L1210 cells attenuated with mitomycin C. OK-432 potentiated syngeneic and semi-syngeneic transplantation resistance in vivo and augmented primary and secondary cytotoxicity mediated by spleen cells in vitro. In vivo administration of ADR and CY together enhanced in vivo tumor transplantation resistance and in vitro cytotoxicity was blocked, but this inhibition was reversed by injection of OK-432. The results suggested that OK-432 acted by increasing the activity of cytotoxic spleen cells against L1210 cells, which are fast-growing and poorly immunogenic, and that this cytotoxicity killed tumor cells that survived the chemotherapy.

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The inhibitory effects of about 30 compounds, mainly pyrimidine and pyridine derivatives, on 5-fluorouracil degradation catalyzed by dihydrouracil dehydrogenase (DHU dehydrogenase) were investigated. The inhibitory activities of 5-substituted uracil derivatives decreased time-dependently during preincubation with liver extracts, indicating that these compounds are substrates of DHU dehydrogenase. During preincubation, 4, 6-dihydroxypyrimidine derivatives were found to be converted to barbituric acid derivatives, which have stronger activities. The inhibitory activity of 2, 4-dihydroxypyridine (3-deazauracil), which was stronger than that of uracil, did not change during preincubation, indicating that this compound is not a substrate but is an inhibitor of DHU dehydrogenase, and suggesting that 2, 6-dihydroxypyridine (1-deazauracil) could be a potent inhibitor. In the light of these findings, we examined various derivatives of barbituric acid, 2, 4-dihydroxypyridine and 2, 6-dihydroxypyridine. Among these compounds, 3-cyano-2, 6-dihydroxypyridine and 5-chloro-2, 4-dihydroxypyridine were the strongest inhibitors with K_i values for DHU dehydrogenase of 2.3×10^-7M and 3.6×10^-7M, respectively.

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Female C3H/HeN mice bearing solid FM3A tumors were treated with a combination of ACNU (or Nimustine hydrochloride) and radiation in order to investigate the antitumor effects and also to determine the optimal treatment schedule of ACNU in such combined therapy. ACNU was ntravenously injected either twice with an interval of 2 weeks and a dose of 30mg/kg (intermittent large-dose treatment), or as 4 weekly doses of 15mg/kg (fractionated small-dose treatment). Local irradiation (5 Gy) at the tumor site was performed twice with an interval of 2 weeks between the doses. Synergistic effects were obtained by the combination of ACNU and radiotherapy in terms of tumor growth inhibition and prolongation of survival. Histology also revealed much greater reduction of viable tumor cells in the case of combination treatment. With either treatment with ACNU alone or in combintion with irradiation, the intermittent large-dose injections resulted in better inhibition of tumor growth than did the fractionated small-dose injections, but the survival times with the intermittent treatments were not as prolonged as would be expected from the direct effects. Loss of body weight and depression of WBC number were also much more severe in the intermittent treatment group. Thus, it may be concluded that when ACNU is combined with radiotherapy, the intermittent large-dose regimen is not necessarily superior to the fractionated small-dose one.

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