The patterns of epithelial proliferation in the urinary bladder during carcinogenesis were examined sequentially in rats given drinking water supplemented with 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) for 12 weeks and then water without BBN for 18 weeks. Animals received 120μg of bromodeoxyuridine (BrdU) per hour continuously via an osmotic minipump for 4 days before sacrifice and labeled cells were detected immunohistochemically using monoclonal antibody against BrdU. Two types of BBN-induced epithelial lesions were distinguishable: reversible changes characterized by proliferation of basal cells only, and irreversible changes with high and irregularly distributed incorporation of label throughout the epithelium. Simple hyperplasia, and papillary or nodular hyperplasia consisted of areas of reversible and/or irreversible changes, whereas papilloma and cancer consisted of areas of irreversible changes.

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Sera and cerebrospinal fluid (CSF) from patients with human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy (HAM) were analyzed by Western blotting, and normal human leukocytes were transformed by co-cultivation with HAM patients' leukocytes. The sera and CSF from all HAM patients formed specific bands with HTLV-1 viral proteins, including p19, p24, p28, p32, p40 and p53. After 2-3 weeks of co-cultivation, scattered foci of cell aggregates were noted on macrophage sheets. Surface markers of the transformed cells were OKT3(+), OKT4(+), OKT8(-), IL-2 receptor (+) and EBNA(-). Chromosome analysis showed a normal karyotype. HTLV-1 viral genome was integrated into DNA isolated from transformed cell lines. Electron microscopy revealed type C virus particles in transformed T-cell lines. These results indicate that peripheral leukocytes from HAM patients can transform HTLV-1-negative leukocytes and HAM patients have the potential to acquire adult T-cell leukemia in the future.

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Soluble interleukin 2 receptors (IL 2R) from human retrovirus-infected cells were analyzed. All the T cell lines integrated with human T cell lymphotropic virus (HTLV)-I and -II, or transfected with HTLV-I gag-pX gene, were found to release high levels of IL 2R constitutively. In contrast, this was not found in T cell lines in which HTLV-I was integrated but not expressed, human immunodeficiency virus (HIV)-infected T cell lines, or T cell, B cell, granulocyte and macrophage cell lines which were HTLV-I negative. These results raise the possibility that the pX-gene product might stimulate the generation of soluble IL 2R. In the sera from patients with adult T cell leukemia, large amounts of IL 2R were released, in contrast to sera from healthy carriers of HTLV-I. The molecular weight of IL 2R was determined to be about 50 KD by size-exclusion HPLC.

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Using polymorphic DNA markers, we compared the constitutional and tumor genotypes of patients with multiple endocrine neoplasia type 2A (MEN2A). We found loss of constitutional heterozygosity at the D22S9 locus in one out of 9 medullary thyroid carcinomas (MTCs). No loss of heterozygosity was detected at 12 other loci in any of the MTCs tested. Loss of heterozygosity at D22S9 and/or D22S1 was also demonstrated in 2 out of 5 pheochromocytomas tested. These results suggest that loss or mutation of a gene on chromosome 22 may play an important role in tumorigenesis in MEN2A.

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In order to analyze the rapidly rising male mortality rate from liver cancer and to estimate the preventive effect of large-scale hepatitis B vaccination, we calculated the future number of deaths from liver cancer and cases of hepatitis B-related liver cancer by the linear/curved regression technique and the simulation method. Published statistics and research data were utilized for the calculation. The results of the calculation showed: 1) the increase in number of deaths from liver cancer for males would continue at the same rate as today, 2) this increase in number of deaths from liver cancer was caused by factor(s) not related to hepatitis B infection, 3) the ratio of hepatitis B virus horizontal infection to vertical infection has been sharply decreasing since the 1970s. The effect of the decrease of hepatitis B virus horizontal infection on the prevention of liver cancer is expected to be larger and to appear at an earlier stage than the effect of large-scale hepatitis B vaccination in Japan.

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The frequency of active ras oncogenes in human bladder cancers associated with schistosomiasis, the cause of which is suspected to be a chemical carcinogen(s) in urine, was examined. Of 9 squamous cell carcinomas of the bladder surgically obtained in Egypt, none scored as positive in the regular DNA transfection assay using NIH/3T3 cells as recipients. The restriction fragment length polymorphism assay at codon 12 of the H-ras gene confirmed the absence of an activating mutation at this site in all of them. Western blotting analysis of electrophoretic mobilities of the ras p21 proteins, a method which can detect at least some of the point mutations within codons 12 and 61 of ras genes, suggested a point mutation within codon 61 in one out of the 7 tumors analyzed. In contrast to the low frequency of detection of mutationally activated ras oncogenes, enhanced expression of the ras p21 proteins was demonstrated in 4 of them by this analysis. The carcinogenic process involved in the endemic bilharzial bladder cancers is thus not associated with detectable point mutations within ras genes at a higher frequency than those in non-bilharzial bladder cancers in Japan or the USA.

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Mouse monocytic Mm-A cell line is a highly leukemogenic variant cell line of the monocytic and non-leukemogenic Mm-1 cell line, which developed spontaneously from mouse myeloid leukemia M1 cells. Growth-inhibitory factor (GI factor) for Mm-A cells was found in conditioned medium (CM) of differentiation inducer-resistant myeloblastic M1 cells (clone R-1). The R-1 cells were cultured with or without 2% calf serum for 2 days, and the CM was fractionated with 50% ammonium sulfate and used as the GI factor preparation (termed R1CM). When Mm-A cells were cultured with 5% (v/v) R1CM for 3 days, their growth was inhibited about 80%. This inhibition of Mm-A cell growth by R1CM was irreversible. This GI factor also inhibited the growth of M1 cells that had been pretreated with inducer and had expressed some differentiation-associated properties but still retained a proliferative capacity. In contrast, it scarcely inhibited the growth of untreated M1 cells. The GI factor inhibited the growth of other mouse monomyeloblastic leukemic WEHI-3B D⁺ cells pretreated with a differentiation inducer, retinoic acid, and mouse monocytic leukemia J774.1 cells. However, it did not affect the growth of human monocytic (U937 and THP-1) or myeloid (KG-1, ML-1, and HL-60) cell lines. These results suggest that GI factor produced by parent myeloblastic and inducer-resistant M1 cells preferentially inhibits the growth of mouse monocytic leukemia cells in intermediate stages of differentiation from myeloblastic leukemia cells to mature macrophages.

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Possible stimulation of the DNA repair capacity by sodium butyrate in normal and xeroderma pigmentosum (XP) cells was investigated. XP cells belonging to the complementation group F showed considerable stimulation of DNA repair by sodium butyrate in terms of both the amount of unscheduled DNA synthesis (UDS) and the colony-forming ability after UV irradiation. UDS in XP cells belonging to the complementation group A was not enhanced, while normal cells showed slight enhancement, but less than that of XP F cells. In XP A, XP C, and normal cells, sodium butyrate treatment enhanced the killing effect of UV irradiation. The residual repair capacity in XP F cells appeared to be stimulated by sodium butyrate.

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The effects of androgen or glucocorticoid on the induction of secretory proteins in SC-3 cells (a cloned cell line from Shionogi carcinoma 115) were examined in a serum-free medium [Ham's F-12: Eagle's minimum essential medium (1:1, v/v) containing 0.1% bovine serum albumin]. Through analysis of [³⁵S]methionine-labeled proteins by one-dimensional (sodium dodecyl sulfate polyacrylamide) gel electrophoresis, we successfully demonstrated the production of a testosterone (10^-9-10^-6M)- or dexamethasone (10^-8M)-induced secretory protein with a molecular weight of 24, 000 in SC-3 cells. However, 17β-estradiol (10^-8M) or progesterone (10^-8M) had no such effect. The production of the secretory 24K protein increased slightly within 2hr after the addition of 10^-8M testosterone and increased further thereafter. The addition of tunicamycin to the incubation mixture with [³⁵S]methionine resulted in the identification of a testosterone-or dexamethasone-induced secretory protein with a molecular weight of 20, 000. By the use of two-dimensional gel electrophoresis, we could demonstrate that both testosterone (10^-8M) and dexamethasone (10^-8M) added to the serum-free medium stimulate the secretion from SC-3 cells of the same 5 proteins (30-40K, 39K, 30K, 27K proteins and the secretory 24K glycoprotein). The present findings show that physiological concentrations of both androgen and glucocorticoid induce the secretion of the same 5 proteins from SC-3 cells.

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The effect f portal venous (pv) administration of syngeneic tumor cells on the potential to generate anti-tumor immune resistance was examined. C3H/He mice were inoculated with syngeneic X5563 tumor cells via the pv or intravenous (iv) route. A single pv administration of 10, 000R X-irradiated X5563 cells into C3H/He mice not only failed to induce anti-X5563 immunity but also abolished the capability of the mice to develop anti-X5563 in vitro cytotoxic and in vivo protective immunity as induced by intradermal (id) inoculation of viable X5563 cells followed by the surgical resection of the tumor (immunization procedure). Such immunosuppression was also obtained by pv inoculation of tumor cells after the above immunization procedure. The immunosuppression induced by the pv injection of tumor cells before or after the immunization procedure was tumor-specific, since the pv sensitizing regimen using X5563 tumor cells did not interfere with the generation of immunity against another syngeneic tumor, MH134. More importantly, the pv route-induced suppression contrasted with the lack of inhibiting effect of iv inoculaton with tumor cells on the induction of the tumor immunity. These results indicate that the host's tumor-specific immune capability is markedly suppressed in an anti-tumor sensitizing stage-independent manner when tumor cells enter the pv circulation.

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A monoclonal antibody, NCC-SG-007, was raised with a formalin-fixed adenoid cystic carcinoma of the salivary gland as the immunogen. The reactivity of the antibody was tested on paraffin sections using the avidin-biotin-peroxidase complex (ABC) method. NCC-SG-007 reacted with 36% of the adenoid cystic carcinomas, 42% of the pleomorphic adenomas and 14% of the mucoepidermoid tumors of the salivary gland which were tested. The antibody also reacted with some normal tissues and various other tumors. Surprisingly, NCC-SG-007 showed a reactivity as high as 94% with gastric carcinomas. The antigenic determinant was revealed to be a carbohydrate chain with no terminal sialic acid, the molecular weight of which was estimated to be 1.5×10⁶ daltons. This antigen differed from other previously reported gastrointestinal cancer-associated antigens. The antibody should be useful for studying adenoid cystic carcinomas of the salivary gland and gastric cancers.

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Monoclonal antibodies reactive to line 10 (L10) hepatocarcinoma cells, but not to L₂C leukemia cells, of strain 2 guinea pig were produced and characterized. Complement-mediated cytolysis assay and quantitative absorption analysis revealed that one of the monoclonal antibodies, 3C4 antibody (IgG2a), was highly specific for L10. Neither normal guinea pig tissues including adult and fetal liver, nor other hepatomas of strain 2 guinea pig, line 1 (L1) and Hepatoma III, were reactive with the 3C4 antibody. Another antibody, 2E4 (IgM), was reactive to the liver, kidney and spleen cells but not to the thymocytes, lymph node cells, brain and red blood cells of the guinea pig. The 2E4 antibody reacted to Hepatoma III but not to L1 cells. Therefore, 3C4 antibody is specific to a tumor-associated antigen of L10 hepatoma, and 2E4 antibody is cross-reactive with a certain differentiation antigen of normal tissues. The 3C4 antibody showed antibody-dependent cytotoxicity. Since 3C4 antibody was different in the isotype and the antigen recognized from a monoclonal antibody (D3) which was reported from the NIH in the USA, 3C4 is a new monoclonal antibody reactive with L10 hepatocarcinoma.

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The expression of Lewis blood group antigens, Lewis^a, Lewis^b, Lewis^x and Lewis^y, in 40 non-cancerous livers and in 20 hepatocellular carcinomas (HCC), 5 cholangiocarcinomas (CC), and 6 combined hepatocellular and cholangiocarcinomas (Comb) was studied by immunohistochemistry using monoclonal antibodies. Although normal hepatocytes did not express any of the four Lewis antigens, Lewis^y expression was detected in some hepatocytes at the periphery of pseudolobules in cirrhotic liver. While Lewis^x in bile duct epithelial cells was undetectable in normal livers, it was detected in some cases with non-cancerous liver diseases. Lewis^x and Lewis^y were detected in 30% of HCC and 100% of CC and Comb. Non-cancerous bile duct epithelial cells in almost all instances were stained strongly by anti-Lewis^a and/or anti-Lewis^b antibody. Proliferating bile ductules within Glisson's sheath and pseudolobules could be clearly detected by Lewis^a and Lewis^b staining. Lewis^a and Lewis^b were never detected in non-cancerous hepatocytes, and were only rarely detected in HCC, but were expressed in most cases of CC and Comb. These results suggested that Lewis^a and Lewis^b are useful markers for differentiation towards biliary epithelial cells in the liver, and that Lewis^x and Lewis^y expression might be associated with states of increased or altered cell proliferation.

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To determine the optimal conditions for clonogenic assay, the antitumor activities of 5-fluorouracil (5-FU) in vitro and in vivo were compared from a pharmacodynamic viewpoint in a human carcinoma xenograft-nude mouse system. In the clonogenic assay, tumor cells were exposed to 5-FU continuously for 2 weeks, and the results of the assay were evaluated in terms of colony survival rates (T/C). Tumor-bearing BALB/c male nude mice were treated with 5-FU, and the results were evaluated in terms of the lowest values of relative mean tumor weights (T/C). To compute the area under the curve (AUC) after administration of 5-FU in vitro and in vivo, agar and serum levels of 5-FU were measured by bioassay. Antitumor activities in terms of T/Cs against a gastric carcinoma strain (H-111) depended highly on the AUCs in vitro and in vivo. The T/C in vitro (z) was correlated with the AUC in vitro (w), z=58.4×0.99331^w, and the T/C in vivo (y) was also correlated with the AUC in vivo (x), y=52.1×0.88552^x. On the assumption that the T/Cs of these two regression equations were equivalent (y=z), a correlation between w and x was derived. To predict the T/C at the maximum tolerated dose of 5-FU in mice, the optimal drug concentration in vitro was calculated to be 2.7μg/ml, which also proved to be suitable for seven other carcinoma strains. Our experimental system was thought to be adequate for selecting the optimal drug concentration in the clonogenic assay.

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A matched case-control study was performed to identify the etiologic factors for thyroid carcinoma in teen-agers. Twenty-seven cases and 69 controls were investigated to assess the significance of various maternal and subject factors. Irradiation during infancy was the only factor which showed a statistically significant association with the incidence of thyroid cancer in teen-agers (summary χ²=8.040; d.f.=1; P<0.005). The estimated dose ranged from 0.2 to 40 rads on the head, neck, or chest during infancy.

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Serial biopsy specimens of gastric lesions of patients in whom cancer was diagnosed at follow-up examination were reexamined retrospectively; the source materials were 17, 429 gastric biopsies carried out on 14, 779 patients from 1971 to 1985 at Aichi Cancer Center Hospital. Among these cases, cancers were found at follow-up in 34 cases at locations distinct from the initially biopsied lesions, and in 41 cases, cancerous changes were detected at follow-up at the locations of the original lesions. These 41 cases amounted to 0.5% of the total biopsied cases with negative results. The average interval from the first to the last endoscopic examination in these 41 cases was 52.1 months and the longest interval was 150 months in the case of a scarred lesion. Based on the reexamination of the histology of the biopsy specimens taken at the first examination, these 41 cases were classified into three subgroups: 1) no precancerous atypical changes were observed, 2) doubtful precancerous changes were observed and 3) minute or slight malignant changes did exist but had been overlooked. The number of cases in the first category was 33 (80.5%), among which ulcer or its scar was most common, the number in the second category was 6 (14.6%), most of them being polypoid lesions, and the number in the third category was 2 (4.9%). Histological features of some cases in each subgroup are presented and the results are discussed.

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