Human T-cell leukemia virus type I has a unique sequence pX and the product p40^x was proposed to be a specific trans-acting transcriptional activator of expression of the viral gene. Recently, a second pX protein p27^x-III in addition to p40^x was identified; these two proteins are encoded by overlapping frames III and IV (x-lor). For determination of which product is the trans-acting activator, site-directed mutations were introduced into the pX sequence which was placed under the metallothionein promoter. On cotransfection with pLTR-CAT (a plasmid containing the LTR of HTLV-I and chloramphenicol acetyltransferase gene), only the mutations that affected p40^x expression inactivated the transcriptional activation from the LTR.

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We found that p19^gag of HTLV-I and p23^gag of HTLV-II are myristylated. The p28, which is immunologically cross-reactive with monoclonal antibody against p19^gagof HTLV-I was also shown to be myristylated in the HTLV-I-infected cell lines MT-2 and HUT102. However, no myristylated p28 was found in HTLV-II-infected cell lines, Mo and Tonl.

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The methylation state of cellular oncogenes (c-oncs) and epidermal growth factor (EGF) receptor gene from human liver tissues was examined by means of restriction endonuclease analysis. c-myc and EGF receptor gene from hepatocellular carcinoma and fetal liver were substantially hypomethylated in comparison with those genes from normal liver, while the extents of methylation of c-mos and c-Ki-ras genes were the same among these tissues. It can be speculated that the specific hypomethylation of c-myc and EGF receptor genes may be associated with the development of hepatocellular carcinoma.

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We have already found that H-2 complex has an apparent effect on the development of lung tumors in mice. In connection with this finding, the incidence of in vivo urethan-induced chromosomal aberrations of bone marrow cells was examined in H-2 congenic strains. These strains could be divided into two groups, one with a higher frequency of chromosomal aberrations such as A.AL (H-2^a1), A.TL (H-2^t1), B10.A (H-2^a) and B10.S (9R) (H-2^t4), and the other with a lower incidence such as B10 (H-2^b), A.SW (H-2^s), B10.S (H-2^s) and B10.S(7R) (H-2^t2). These results suggested that a putative gene mapped to the chromosomal segment between Eβ and D may affect the incidence of urethan-induced chromosomal aberrations.

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To obtain definitive evidence that milk-borne infection plays a critical role in the endemy or mother-to-child transmission of human T-cell leukemia virus type-I (HTLV-I), we inoculated concentrated fresh human milk cells obtained from HTLV-I carrier mothers into the oral cavity of a common marmoset (Callithrix jacchus). Twenty-eight milk samples were collected (5-10ml each) from 17 carrier mothers in the first week after delivery. Cells in the milk were centrifuged down and resuspended in 1/10vol of the milk fluid. The concentrated cell suspensions were successively inoculated into the oral cavity of a common marmoset. The marmoset was found to be seroconverted by indirect immunofluorescence assay at 2.5 months after the first inoculation of the milk (3.5×10³cells in total), and was later confirmed to be infected with HTLV-I by the detection of viral antigen expression in short-term cultures of its peripheral blood T-lymphocytes. The results strongly support the working hypothesis that milk-borne infection plays a significant role in the mother-to-child transmission of HTLV-I.

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Seryl/threonyl-protein kinases in cytosolic and particulate fractions from rat liver and AH-13, a rat ascites hepatoma, have been studied by chromatographing these fractions on DEAE-cellulose and assaying the eluates with casein, phosvitin, histone and protamine as substrates. Liver cytosolic fraction contains a group of well-characterized seryl/threonyl-protein kinases, namely, casein kinases I and II and histone kinases I and II. Liver particulate fraction, on the other hand, is almost totally devoid of casein kinase I and histone kinase I but contains an additional peak of casein kinase tentatively designated casein kinase III. In AH-13, cytosolic casein kinase I is markedly increased and particulate-associated casein kinases II and III are moderately increased as compared with liver. Moreover, it was found that in AH-13, the histone kinase I level is high in the particulate fraction but markedly decreased in the cytosolic fraction. It is suggested that particulate-associated histone kinase I may be of cytosolic origin.

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Concentrations of creatine kinase (CK) B subunit (CK-B) in tumor tissues and in sera of patients with various lung carcinomas were determined, together with the concentrations of neuron-specific γ-enolase (γ subunit of αγ and γγ enolases), by the use of a sensitive enzyme immunoassay method. The CK-B and γ-enolase levels were enhanced in tissues of small cell carcinoma of the lung. The average tissue contents of CK-B in small cell carcinoma (SCCL), adenocarcinoma (ADCL) and squamous cell carcinoma (ECCL) of the lung, and normal lung were 2320, 308, 163, and 372ng/mg protein, respectively. The contents of γ-enolase in those tissues were 1460, 276, 225, and 42.7ng/mg protein, respectively. Serum CK-B concentrations in healthy adults (n=100) were 0.53±0.22ng/ml and ranged from 0.25 to 1.44ng/ml, but they were significantly increased (>1.5ng/ml) in some patients with SCCL (26/42 cases, 62%), ADCL (7/36, 19%), ECCL (7/37, 19%), and large cell carcinoma of the lung (LCCL, 4/13, 31%). Serum CK-B was also enhanced in some patients with breast carcinoma and in a few cases in carcinomas of the stomach, colon and pancreas. Serum concentrations of CK-B were well correlated with those of γ-enolase in patients with SCCL (r=0.667, n=83, P<0.01) and LCCL (r=0.689, n=20, P<0.01), but poorly in patients with ADCL and ECCL. Since serum CK-B concentrations in patients with SCCL changed in parallel with the clinical course during treatment, serum CK-B may also be a useful biomarker, as well as neuron-specific γ-enolase, for monitoring the clinical course of patients with SCCL.

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Ca²⁺-activated, phospholipid-dependent protein kinase (protein kinase C) activities in human gastric mucosa and carcinoma were examined. The bulk of protein kinase C activity was associated with the cytosol in both gastric mucosa and carcinoma. Protein kinase C activity in the soluble fraction in human gastric carcinoma was significantly higher than that in nonneoplastic gastric mucosa (P<0.05), the values being 357.7 and 215.3units/g protein, respectively. On the other hand, cAMP-dependent protein kinase (protein kinase A) activity was decreased slightly in gastric carcinomas. The ratios of protein kinase C to protein kinase A in human gastric carcinoma and nonneoplastic mucosa were 0.849 and 0.435, respectively, the difference being significant (P<0.01). In DEAE-cellulose column chromatography, the elution profiles of protein kinases from gastric carcinomas were the same as those from gastric mucosas except that a larger peak of protein kinase C was found. It seems probable that increased protein kinase C activity plays an important role in the growth of human gastric carcinoma as a signal transducer.

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A comparative study has been made on the levels of vitamins A and C in normal and malignant conditions in human and murine subjects. Further, the effect of supplemental vitamins A and C on tumor take, host-survival and tumor growth have been studied in a number of transplantable and induced tumors in mice. The vitamins were assayed in sera samples from normal subjects, patients with cancer of the uterine cervix or ovary, and leukemia and lymphoma patients. Among the murine group the tumors included sarcoma 180 in solid and ascitic form, benzo[a]pyrene-induced fibrosarcoma, Dalton's ascitic lymphoma and Schwartz lymphoblastic leukemia. The serum level of vitamin C was found to be lower than that of the normal controls in all cases studied. The level of vitamin A was found to be higher in cancer patients in the human group and lower in the murine group when compared with their normal controls. Studies on murine tumors showed that supplementary vitamins administered at the initial phase of tumor development reduced the percentage tumor take and the rate of tumor growth, and improved host survival, indicating that these vitamins have a protective role in the murine system.

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We determined the properties of three adriamycin (ADM)-resistant cell lines isolated from a clone of herpes simplex virus (HSV) type 2-transformed Syrian hamster cells. The ADM-resistant lines (ADM^R-6, -7 and -9) established by continuous exposure of the cells to ADM were 20 to 30 times more resistant to ADM than was the parent line. These resistant lines exhibited cross-resistance to daunomycin, actinomycin D, chromomycin A3 and vincristine, but not to mitomycin C, bleomycin or methotrexate. Uptake and efflux studies with [³H]ADM indicated that one line (ADM^R-9) incorporated and retained lesser amounts of [³H]ADM than did the parent line but the other two lines accumulated and retained as much of the drug as did the parent line. Expression of cell surface antigens, detected by immunofluorescence tests with antiserum to HSV type 1, was at a low level (about 8% positive) in the parent line after replating of the cells, although it was enhanced by treatment of the cells with ADM (0.25μg/ml). However, the antigens were strongly expressed (_??_35% positive) on the surface of cells of the three resistant lines in the absence of ADM. Both antigen expression and ADM resistance of the cells were relatively stable in in vitro cultivation but were unstable in in vivo cultivation, suggesting that the constitutive expression of the antigens is closely associated with the phenotype of ADM resistance.

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A combined hepatocellular and cholangiocellular carcinoma of diffuse type in a Japanese man is described. A small localized solitary tumor apparently grew rapidly into a diffuse-type carcinoma, and the liver weight increased about 4-fold during the last two months. The clinical course of this case was as expected for a diffuse type of hepatocellular carcinoma except that unusually high levels of serum carcinoembryonic antigen were found. The patient died of hepatic failure with systemic bleeding five months later. At autopsy, multiple small nodules were suspected to be intrahepatic metastatic foci because portal tumor thrombus was observed in the right antero-superior segment where the initial tumor was localized. Histologically, the tumor had components of both hepatocellular and mucin-producing cholangiocellular carcinoma. This is believed to be the first report on a diffuse type of combined hepatocellular and cholangiocellular carcinoma initially presented as a localized small nodule.

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The mechanism of murine tumor necrosis factor (TNF) cytotoxicity against tumor cell lines (L929, HeLa, K562) was investigated. Electron microscopic observation revealed that most of the organellas of L929 cells incubated with partially purified murine TNF underwent almost complete lysis with no drastic disruption of the cytoplasmic membrane, while injection of the TNF into the cytoplasm or nuclei of L929 cells caused no apparent morphological change or growth inhibition. Preincubation of the TNF with tumor cells (L929, HeLa, K562) resulted in a decrease in cytotoxic activity which was proportional to their susceptibility to TNF, thus indicating their absorption of TNF. The susceptibility of L929 tumor cells to TNF was apparently suppressed by treatment with proteases, suggesting the existence of protease-sensitive recognition sites for TNF on the tumor cell.

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Fischer 344 (F344) rats hyperimmunized with syngeneic 9L/R₃ glioma cells produced antibody selective to glioma cells. Hybridomas prepared from the spleen cells of the immunized rat were cloned, and we obtained a hybridoma clone which produced monoclonal IgM antibody, termed FR77, that showed selectivity to glioma cells. Immunoperoxidase staining of cultured cells revealed that FR77 was reactive with 3 lines of rat glioma cells but not with normal F344 rat fibroblasts. Immunohistochemical staining of F344 rat tissue sections with biotinylated FR77 demonstrated that FR77 could bind with glioma tissue developed by intracerebral injection of 9L/R₃ glioma cells but not to normal parts of the brain tissues and other normal tissues tested. The FR77-defined antigen was observed to be mainly localized in cytoplasm of glioma cells but a small portion of the antigen was also detected on the glioma cell surface.

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The antigen (YH206 antigen) corresponding to the monoclonal antibody (MoAb) YH206 (IgM), which was produced against a lung adenocarcinoma cell line A549, was found to be an extremely high-molecular-weight protein of over 330, 000 daltons by means of SDS-PAGE and Western blot analysis. The immunohistological distribution of the antigenic determinant recognized by MoAb YH206 was found to be limited to adenocarcinoma tissues. It was shown by reversed passive hemagglutination assay (RPHA) that the antigen could be detected not only in the spent medium from human lung cancer cell line A549 but also in the sera from cancer patients. Only 3 out of 30 (10%) healthy donors and 4 of 31 (12.9%) patients with benign diseases had serum antigen levels of more than 1/64 dilution. In contrast, 42 of 87 (48.3%) patients with lung cancer and 29 of 50 (58.0%) patients with cancers of the digestive organs had elevated levels of the antigen. As regards the relation between antigen levels and clinical stages of lung cancer, values of more than 1/128 dilution were detected only in patients with stage III or IV disease.

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In connection with autologous bone marrow transplantation (BMT) in null cell-type acute lymphocytic leukemia (Null-ALL) patients, the optimal conditions for in vitro elimination of leukemia cells from transplanted bone marrow cells with monoclonal antibodies (MoAb) plus rabbit complement (C) were investigated. Three monoclonal antibodies, NL-1, NL-22 and HL-47, which were produced in our laboratories and shown to react predominantly to Null-ALL cells, were used. In most cases, 10μg/ml of MoAb and 50% of C were required for optimal killing of leukemia cells. Combined use of MoAbs and multiple treatments were studied, but generally no significant increase in the cytotoxicity to the target leukemia cells was observed as compared with a single treatment with the most effective single MoAb. Selection of C was very important, because some lots had lower C activity from the beginning or after incubation with normal bone marrow mononuclear cells. Nonspecific cytotoxicity to human hematopoietic cells was also observed in some lots of C. Under the optimal conditions thus defined, the three MoAbs did not inhibit the growth of granulocyte-macrophage colony-forming units (CFU-GM) or three other types of hematopoietic stem cells. Modulation of the antigens was also examined, and NL-1 antigen showed mild modulation, whereas NL-22 and HL-47 antigen did not. The results obtained indicated that these three MoAbs may be useful for eliminating Null-ALL cells in vitro from bone marrow in autologous BMT.

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Autologous bone marrow transplantation (BMT) in null cell-type acute lymphocytic leukemia (Null-ALL) was carried out after depletion of leukemia cells from transplanted bone marrow. Patients' autologous bone marrow cells were harvested during remission and treated in vitro with complement and three monoclonal antibodies (NL-1, NL-22 and HL-47) reactive to Null-ALL cells, and then cryopreserved. Three patients were transplanted with the antibody-treated bone marrow cells during the first remission period after preconditioning with intensive chemotherapy and total body irradiation, while transplantations in two other patients, who were in poor clinical condition, were done during the fourth remission period and the third relapse, respectively. Good preservation of hematopoietic stem cells after antibody treatment and cryopreservation of bone marrow cells was demonstrated in all five cases studied. Clinically, prompt recovery of white blood cells and platelets was observed in the three patients who received BMT during the first remission period; two of them have continued remission (2 and 15 months), while the other relapsed after 7 months of remission. These results suggested that autologous BMT with these three antibodies may be an effective mode of therapy for Null-ALL patients.

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In 8 postoperative patients with gastric cancer the effectiveness of fat emulsification of tegafur as a means of improving the drug distribution in the lymphatic tissue was studied. A water-in-oil type of emulsion of tegafur (FT-w/o) and an oil-in-water type of emulsion of tegafur (FT-o/w) were orally administered. A fistula in the thoracic duct, prepared in advance, was used to collect lymph. The concentrations of tegafur and 5-FU were compared in simultaneously obtained specimens of lymph and peripheral blood. In the case of FT-w/o, the tegafur concentration at 30-60min after administration was significantly higher in both the lymph and plasma than that found using FT-o/w. In the case of FT-w/o, the 5-FU concentration at 30-120min after the administration was significantly higher than that found with FT-o/w in both the lymph and the plasma. Thus, FT-w/o shows excellent distribution in both lymph and blood, and is considered useful as an adjuvant chemotherapeutic agent in the postoperative treatment of gastric cancer patients.

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Primary radiation-induced or radiation leukemia virus (RadLV)-induced T-leukemias/lymphomas were treated in vivo in an early to advanced state by using 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU was given at various times after the tumor induction procedure. Death from RadLV lymphomas which had been initiated in 33±3 day old C57BL mice by intrathymic injection of RadLV was scored in untreated, BCNU-treated or BCNU and cellular adjuvant treated mice. Intrathymic RadLV injection in 33±3 day old mice produced tumors in 98% of injected mice. Median survival time (MST) was increased by BCNU and by BCNU plus bone marrow cell therapy whether done 33 or 47 days after RadLV. There was increase in MST from 108 days to 171 days by BCNU and bone marrow cell therapy given 33 days after tumor initiation and to 195 days when therapy was given 47 days after initiation. In radiation-induced lymphomas produced by 190 rad every week ×4 of 33±3 day old mice, spleen cell (×1) therapy or BCNU treatment increased the MST of treated mice from 142 days to 177 days after iv spleen cells or to 195 days after iv-ip spleen cells, and this protocol produced 31% long-term cures. Cellular adjuvant therapy combined with BCNU chemotherapy was effective for curing the lymphomas but cellular adjuvant therapy alone was also highly effective for therapy.

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