A new strain of Epstein-Barr virus was isolated from an epithelioid hybrid cell line derived from nasopharyngeal carcinoma which had been cultured for 14 days at 32°. This virus can transform human cord blood lymphocytes and induce early antigens in Raji cells.

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Summation and synergism in the effects of three tumor promoters on urinary bladder carcinogenesis initiated by a 4-week treatment with 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in male F344 rats were examined. In experiment 1, the sequential administration of sodium saccharin (SS, 5.0%), DL-tryptophan (Tr, 2.0%) and sodium L-ascorbate (SA, 5.0%) in the diet, each for 10 weeks, significantly increased the incidence and the number of bladder tumors over that observed after SS alone or SS followed by Tr. In experiment 2, the simultaneous dietary administration of 2.5% SA, 1.0% butylated hydroxyanisole and 0.01% allopurinol for 32 weeks significantly in. creased the yield of bladder tumors. Paired combinations of promoters or each of the promoters administered alone were associated with a less pronounced promotive effect than when all three were combined. Thus, it is evident from the results of the present investigation that whatever the mechanisms underlying promotion by the different agents, they are capable of working in an additive fashion, under conditions of summation (consecutive administration) or synergism (simultaneous administration).

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On the basis of paradoxical concanavalin A (Con A) staining, the tendency of tumor cells of gastric phenotype to shift to intestinal phenotype and the stability of the latter phenotype in stomach tumors of different sizes were examined quantitatively with an image processor. Phenotypic expression of tumors of the small intestine was also studied. One hundred male Wistar rats were given N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at 50μg/ml in their drinking water for 20 weeks (group 1). Twenty male F344 rats were given methylnitrosourea (MNU) at a dose of 50mg/kg ip twice a week for 2 weeks (group 2). Rats in group 1 were killed in week 50 of the experiment and rats in group 2 were killed in week 25. In group 1, the percentage areas of intestinal-type cells in small, medium and large adenocarcinoma of the stomach were 0.5, 2.7 and 6.6%, the differences between these values being significant (P<0.05-0.01). Intestinal phenotypic expression of tumor cells of the stomach is stable and the proportion of intestinal-type cells in adenocarcinomas of the stomach is higher in the larger tumors. Adenomatous hyperplasias and adenocarcinomas of the small intestine in groups I and 2 were all composed entirely of cells of the intestinal type. These results suggested that intestinal-type cells in adenocarcinoma of the stomach did not originate from intestinal metaplasias but from gastric-type cells in stomach adenocarcinomas.

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Male Donryu rats and B6C3F₁ mice were treated with dietary 3-methozy-4-aminoazobenzene (3-MeO-AAB) (0.06% or 0.09%), and subcellular fractions of the liver were examined for ability to mutagenically activate 3-MeO-AAB and 2 amino acid pyrolysate components using Salmonella typhimurium TA 98 as a tester strain. The treatment resulted in striking induction of enzyme(s) for the mutagenic activation of these aromatic amines in rats, but the effect was smaller in mice. The enzyme(s) involved in the reaction was characterized as an isotype of microsomal cytochrome P-448.

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One of the quinoline-DNA base adducts, QG_I, formed in cells treated with 4-nitroquinoline 1-oxide, was readily prepared in vitro from GMP or dGMP and 4-hydroxyaminoquinoline 1-oxide in the presence of ATP, L-serine, and seryl tRNA synthetase. Synthetic seryl-AMP could be substituted for the enzymatic activation system for QG_I formation. Chemical and spectral analyses of the adduct thus prepared revealed that QG_I can be formulated as N⁴-(guan-8-yl)-4-aminoquinoline 1-oxide, the structure of which is identical with the modified base structure involved in the deoxyguanosine-quinoline adduct, dGIII (nomenclature of Loucheux-Lefebvre et al.) obtained by the chemical modification of deoxyguanosine with monoacetyl and diacetyl derivatives of 4HAQO.

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Various human leukemoblastoid cell lines in logarithmic growth were incubated for 16hr with 0.5μM [³H]fluorodeoxyuridine (FdUrd) and the incorporation of (³H)-FdUrd into DNA was measured. T-acute lymphoblastic leukemia (T-ALL) cell lines incorporated 2.4-7.0 times more [³H]FdUrd into newly synthesized DNA than cell lines from B-lymphoid, pre B-ALL, null cell ALL and acute myeloid leukemia cells. T-ALL cells incorporated 1.93-3.15 pmol of [³H]FdUrd nucleotides into DNA per 10⁷ cells. The amount of [³H]FdUrd incorporated into DNA was inversely correlated with the activity level of uracil DNA glycosylase of the cells. On the other hand, no correlation was observed between the incorporation and the level of deoxyuridine triphosphate diphosphohydrolase of each cell line. However, the amount of FdUrd incorporated into DNA could not be correlated with the antiproliferative activity of FdUrd against these cell lines.

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Dialyzed urine of mice bearing leukocytosis-inducing fibrosarcoma stimulated granulocyte colony formation in semisolid agar culture of human bone marrow cells. Removal of phagocytic cells prior to stimulation did not interfere with the formation of these colonies in the culture. On the other hand, macrophage colonies were predominantly produced when murine bone marrow cells were stimulated by the dialyzed mouse urine. The activity of colony-stimulating factor (CSF) in the urine of normal mice was less than 1/100 of that in the urine of tumor-bearing mice. DEAE-cellulose column chromatography separated the activity stimulating human granulocyte colony formation from that stimulating murine macrophage colony formation. Further purification showed that a sialoglycoprotein with an apparent molecular weight of 80, 000 corresponded to the macrophage CSF, which was devoid of activity toward human cells. The molecular properties of the human-active granulocyte CSF could not be studied further, because it was quite unstable.

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The clonal origin of radiation-induced thymic lymphoma was studied in mice with cellular mosaicism for phosphoglycerate kinase (PGK). Repeated whole-body X-irradiations (4 doses, 1.7Gy each) with intervals of 7 days resulted in development of thymic lymphomas in the mosaic mice. PGK from all lymphomas gave only a single spot on electrophoresis. The results demonstrate the single cell origin of the thymic lymphoma.

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Optimal conditions for the in vitro production of cytotoxic lymphocytes against autologous lung cancer cells were studied. In the time-course experiments, fresh lymphocytes did not exhibit any cytotoxicity against autologous tumor cells, but, when cultured in the presence of T cell growth factor (IL2), the lymphocytes became cytotoxic against autologous tumor cells 3 days after the initiation of incubation and the cytotoxicity continued to increase, reaching a maximum at day 7. The study, conducted to compare the effects of IL2 and phytohemagglutinin (PHA), demonstrated that although lymphocytes cultured with PHA did exhibit a significant cytotoxicity, lymphocytes cultured with IL2 showed much higher activity. Peripheral blood, regional lymph node (RNL) and distal lymph node lymphocytes, and tumor infiltrating lymphocytes were tested as sources for the production of cytotoxic lymphocytes. Among these 4 sources, RNL proved the most consistently reliable source of cytotoxic lymphocytes. The results of in vitro stimulation by autologous tumor cells were ambivalent. Twenty-five experiments that compared in vitro stimulation by tumor cells and no stimulation revealed that 9 cases (36%) showed enhancement of cytotoxicity after stimulation with tumor cells, 5 cases showed inhibition, and the remainder no effect. Characterization of lymphocytes by using monoclonal antibodies indicated that cells lytic for autologous tumor cells are OKT3+, OKT8+, OKT4-, OKM1-.

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A microplate leukocyte adherence inhibition (micro-LAI) assay was performed with peripheral blood mononuclear cells obtained from patients with hepatoma and control subjects (including healthy donors and patients with other diseases). Cell extracts of human hepatoma cells (HCC-M) and human hepatic cells (Chang liver cell) in tissue culture were prepared by sonication followed by centrifugation. The supernatants of these two cell lines were used as a specific antigen and a nonspecific antigen, respectively. It was found that monocytes were major indicator cells and that monocytes produced an LAI reaction in the absence of lymphocytes. Therefore, a repeated microplate monocyte adherence inhibition (MAI) assay was developed, in which the monocyte population of adherent cells is increased by removing nonadherent cells after an initial assay in fetal calf serum-containing medium without test antigens, and monocytes are counted selectively as peroxidase-positive cells in a subsequent second assay with test antigens. With regard to sensitivity and reproducibility, the repeated micro-MAI assay is superior to a micro-MAI assay in which the initial assay is omitted although monocytes are selectively counted. With this simple and sensitive technique a hepatoma-associated immune response to the extract of HCC-M was detected in 16 out of 22 patients (73%) with hepatoma, whereas the false-positive rate was 7% (3/41) in all control subjects.

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Expression of heterophile Hanganutziu-Deicher (HD) antigen on the cell surface of various human malignant tumor tissues was studied by membrane immunofluorescence staining with chicken antiserum against N-glycolylneuraminyllactosylceramide (HD3) and fluorescein-conjugated rabbit anti-chicken IgG. The HD antigen was demonstrated in samples from 38 of 77 (38/77, 49%) cases, consisting of gastric (9/16), breast (8/14), colorectal (3/12), nasopharyngeal (4/7), and uterine (2/3) carcinomas, leukemias (5/10), malignant lymphomas (2/5), and other cancers (5/10). Histological examination indicated that expression of the antigen by the gastric, colorectal and breast carcinomas and leukemias was not related to their histological types. The cytoplasm and the surface of the malignant glandular epithelial cells were both specifically stained by immunofluorescence staining of frozen sections in one colon adenocarcinoma. Glycosphingolipids (GSLs) were extracted from the colon cancer tissue and two HD antigen-active GSLs were detected on thin-layer chromatography (TLC) by enzyme-immunostaining using affinity-purified chicken anti-HD3 and horseradish peroxidase-conjugated rabbit anti-chicken IgG. One migrated to the same position as HD3 on TLC, and the other to a position similar to that of authentic N-glycolylneuraminylneolactotetraosylceramide.

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Correlation studies between the sensitivity of tumor cells determined by clonogenic cell assay and the clinical effect of chemotherapy were performed in patients with primary lung cancer. Thirty-four of 48 patients showed adequate colony foration (_??_30 colonies) to test in vitro chemosensitivity. Colony formation of tumor specimens did not differ with regards to prognostic factors such as sex, age, stage, performance status, and prior chemotherapy. The two criteria of in vitro chemosensitivity employed were greater than 70% and 50% reduction in colony numbers. When in vitro chemosensitivity was defined as a colony reduction of greater than 50%, the true positive rate in the assay was 57%, while the true negative rate was 85%. In this case, the predictive accuracy was 79%. When in vitro chemosensitivity was defined as a colony reduction of greater than 70%, the true positive rate for the assay was 100%, while the true negative rate was 81%. In this case, the predictive accuracy was 82%. By Fisher's exact probability test, the overall in vitro/in vivo correlation was statistically significant (P=0.042<0.05) with the 50% cut-off point, but was not significant (P=0.053>0.05) with the 70% cut-off point.

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