A DNA renaturation method in gel was used to detect amplified DNA fragments in eight gastric cancer cell lines, KATO-III, OKAJIMA, SCH, MKNI, MKN7, MKN28, MKN45 and MKN74 cells, and tissues of three metastases of gastric cancers to lymph nodes. There were amplified DNA sequences in three gastric cancer cell lines. Judging from the intensities of the bands, HindIII-digested DNA fragments were amplified several hundred times in the KATO-III and MKN7 cell lines. DNA from OKAJIMA cell line contained multiple bands with less intensity. KATO-III cells were found to contain a homogeneously staining region in chromosome 11.

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The reversibility of forestomach lesions induced in rats by butylated hydroxyanisole (BHA) was examined. F344 rats were given BHA for 24 weeks, followed by the basal diet, and their forestomach lesions at weeks 24 and 96 were compared histopathologically. Hyperplasias and papillomas showing upward proliferation were found in week 24 but not in week 96. However, downward proliferation of basal cells persisted after the discontinuation of BHA administration. This finding suggests that downward growth of basal cells is not reversible and is important in the development of BHA-induced forestomach tumors in rats.

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The numbers of neutrophils and platelets and the serum calcium (Ca) levels were examined in 12 patients with lymphomatype adult T cell leukemia (ATL) and 35 patients with leukemic-type ATL. The same parameters were also studied in 17 patients with non-Hodgkin's lymphoma and 22 patients with acute lymphocytic leukemia as controls. The numbers of neutrophils and the serum Ca levels were significantly higher in leukemic-type ATL (P<0.01) and lymphoma-type ATL (P<0.05) than in the controls. However, no statistically significant correlation between the numbers of neutrophils and the levels of serum Ca in ATL was observed. Thus, these two abnormal clinical features might be caused by different humoral factors.

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A cell line, SCC-3, was established from the pleural fluid of a patient with non-Hodgkin's lymphoma. Interleukin-2 receptor (IL2-R) was found to be expressed on the cell surface by marker analysis, and morphological, cytochemical, and other marker analyses suggested a monocytic lineage. SCC-3 may be useful for studies on the role of IL2-R in monocytic cells.

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Recombinant human granulocyte colony-stimulating factor (G-CSF) was prepared and its granulopoietic effects on mice were examined. When mice were injected intraperitoneally with the G-CSF daily for 14 days, marked granulocytosis occurred in the mice. The number of progenitor cells (CFU-C) was remarkably increased in the spleen. The results suggest that G-CSF plays a central role in granulocyte production in vivo.

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The carcinogenicity of chrysazin (1, 8-dihydroxy-9, 10-anthracenedione) was examined by dietary administration to C3H/HeN mice. All of the effective mice (17) which were given 0.2% chrysazin diet and which survived more than 500 days developed adenomatons hyperplasia with cystic glands of the cecum. Similar lesions were also seen in the colon of mice in this group. These intestinal lesions were not obtained in any effective mouse (19) of the control group. The incidence of hepatocellular carcinoma of mice given chrysazin (4/17) was significantly higher than that of the controls (0/19). These results indicate that chrysazin is carcinogenic in mice as well as in rats. Some mechanistic aspects of the causation of these intestinal lesions and liver neoplasms are also discussed.

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Suitable conditions for inducing experimental yolk sac tumor (YST), choriocarcinoma and teratomas by simple fetectomy were studied in the rat. Tumors were inducible only when fetectomy was performed between the 7th and 14th gestational day. No YST was induced by fetectomy performed after the 15th gestational day. RT1 (major histocompatibility complex of rat) compatibility was also required for the YST induction and no tumor was induced in RT1-incompatible combinations. Only 39% of fetectomized rats produced YST after mating between an inbred strain and their backcrossed F1 hybrid. Mean survival time of the tumor-bearing rats fetectomized on the 10th gestational day was 116 days. The mean diameter of the tumor was maximum in the group fetectomized in the 9th gestational day, and minimum in the group fetectomized on the 14th gestational day.

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Spontaneous neoplastic and non-neoplastic lesions in 95 male and 96 female Donryu rats which were observed up to 120 weeks of age, were examined. The incidence of spontaneous tumors was 73.7% in males and 88.5% in females. In males, the most frequent tumors were pituitary adenomas, followed by pheochromocytomas and insulomas. In females, uterine adenocarcinomas, mammary fibroadenomas and pituitary adenomas were the most common. Other tumors with relatively high incidences in both sexes included cortical adenomas of the adrenal gland, histiocytic sarcomas of the hematopoietic organs and granular cell tumors of the brain. Various tumors were also found in many other organs and/or tissues, although their incidences were low. The organ distribution and incidences of spontaneous tumors observed in Donryu rats were different from those in other strains of rats such as the ACI, Wistar, F344 or Sprague-Dawley strains. The main non-neoplastic lesions were observed in the lung, cervical lymph nodes and kidney of both sexes. In addition, lesions were also observed in the urinary bladder, prostate and peripheral nerves (spinal nerve roots and peripheral nerves) and/or femoralis muscle of males. Histologically, the most characteristic lesion was radiculoneuropathy with degeneration of the peripheral nerves.

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In order to establish the frequency of human papillomavirus (HPV) infection in cervical precancerous lesions of Japanese patients, cervical materials routinely biopsied in the past year were examined immunohistochemically for the papillomavirus genusspecific antigen. Of a total of 832 cervical biopsy specimens, 46 (5.5%) were immunohistochemically positive for HPV. In this study, 206 patients were diagnosed as having dysplasia or carcinoma in situ (CIS), and HPV antigen was found in 15% of these patients. It was found in 13% of patients with mild dysplasia, 28% of those with moderate dysplasia, 17% of those with severe dysplasia and 4.5% of those with CIS. However, HPV antigen was detected only in the epithelium with dysplastic change, not in cancerous areas.

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A survey of epidermodysplasia verruciformis (EV), a skin disease caused by human papillomavirus (HPV), was made by means of a questionnaire sent to 92 university hospitals. Replies were obtained from 68 hospitals (74% recovery) reporting 66 patients. Fewer patients were reported from north-eastern Japan than from south-western Japan. Many EV patients were from families of consanguineous marriages (44%), showing a high incidence of intra-familial onset after consanguineous marriages. The complication of malignant tumors was observed in 36 of 62 cases (58%). Malignant tumors of the skin developed at an early age in EV patients. These cancers developed predominantly in exposed areas of the skin (72%). The time from onset of skin lesions to the onset of cancer seemed to be related to the nature of the lesions in EV patients. These findings suggest that the interaction of HPVs, ultraviolet rays and host factors is associated with the development of skin carcinomas.

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The DNA of a colon carcinoma-derived cell line (KMS-4) and that of skin fibroblasts from a familial polyposis coli patient were transfected into NIH3T3 cells in order to detect oncogenes associated with the disease. No transformation was observed with the normal skin fibroblast DNA, while the KMS-4 cell DNA was able to transform NIH3T3 cells. Through hybridization with known oncogene probes, the KMS-4 transforming gene was found to be a human activated c-K-ras 2 oncogene. Sequence analysis of the molecularly cloned KMS-4 c-K-ras 2 oncogene showed a single nucleotide transition from G to T at the 12th codon. This results in substitution of cysteine for glycine at this position. On using labeled synthetic oligonucleotides to detect the mutation in codon 12, we found the G to T transition in colon carcinoma cells. This suggests that activation of the c-K-ras 2 oncogene could be associated with colon carcinoma induction.

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Radiobiologically hypoxic fractions of 4 experimental tumors (RIF1, B16, EMT6/KU, SCCVII) of various sizes implanted subcutaneously (sc) or intradermally (id) were compared under identical conditions by using the paired survival curve assay method. The two survival curves for tumors in air-breathing and asphyxiated mice were almost parallel for EMT6/KU and SCCVII tumors, but not for RIFT and B16 tumors. Therefore, hypoxic fractions were estimated by fitting the best parallel lines to the two sets of survival data. The values for 10mm-diameter se tumors in the hind legs of syngeneic mice were 4.7% for RIFT, 4.5% for B16, 14% for EMT6/KU, and 8.5% for SCCVII. The variation of the values is quite small in spite of the variety of biological characteristics of these tumors. Histological examination revealed that EMT6/KU and B16 tumors contained large necrotic areas, while SCCVII and RIF1 had small areas of necrosis. Thus, the hypoxic fraction and the proportion of necrosis in the histological specimens were not clearly correlated. The values for 6mm and 16mm sc EMT6/KU tumors were 5.9 and 22%, respectively, while that for 6mm id tumors was 16%. The values for 5mm and 18mm sc SCCVII tumors were 0.86 and 8.4%, respectively. These results indicated that: (1) id EMT6/KU tumors had a higher hypoxic fraction than sc ones of the same size; (2) the hypoxic fraction increased with increase in tumor size in EMT6/KU tumors, while it reached a plateau at a certain size in SCCVII tumors.

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Monoclonal antibodies HBJ127 and HBJ8, raised against T24 human bladder cancer cells, predominantly react with the cells in proliferating stages and with a portion of epithelial tumor cells, respectively. To investigate the in vivo localization of these monoclonal antibodies, the antibodies were labeled with radioiodine and indium-111 (¹¹¹In) and injected into nude mice transplanted with human bladder tumors. The BT-11 bladder tumor had the highest concentration of radioiodinated HBJ127 and HBJ8 monoclonal antibodies, with 11.6 and 14.3% of the injected dose per gram and with a tumor-to-blood ratio of 2.6 and 1.6, respectively, at 4 days after the administration. An irrelevant monoclonal antibody did not show any specific accumulation in the BT-11 tumor. The ¹¹¹In-labeled HBJ127 antibody was also localized in the tumor with a higher tumor-to-blood ratio than the radioiodinated antibody. The xenografted BT-11 tumor was successfully visualized with the radiolabeled HBJ127 and HBJ8 antibodies by scintigraphy. These monoclonal antibodies and the human bladder tumor xenografts may provide a good model for radioimmunoim aging and possibly therapy.

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Six murine monoclonal antibodies (mAbs) reactive with carcinoembryonic antigen (CEA) were prepared. Four of these, CA204, CA205, CA206 and CA208, were specific to purified CEA whereas the other two, NA201 and NA203, were also reactive with the non-specific cross-reacting antigen (NCA). These mAbs were all IgGl, except one IgG2a (CA206), with high affinities for CEA. NA203, CA204 and CA208 appear to define three different epitopes on the CEA molecule as determined by competitive binding assay. These mAbs also reacted with CEA-producing cells. The treatment of the cells with tunicamycin did not affect the binding of the mAbs to CEA-producing cells. None of the above mAbs bound to CEA-related antigens, NCA-2, α₁-acid glycoprotein, or blood group antigens. The combination of CA208 (solid phase) and CA204 (tracer) was used to construct a sensitive CEA-specific sandwich ELISA to detect CEA in the sera of patients with various malignant and non-malignant diseases. Particularly when CEA values were low in sera from non-cancerous patients, the above mAbs sandwich assay showed reduced background reactivity with NCA-like substances and permitted the detection of CEA at a level as low as 1ng/ml.

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Cytotoxic T lymphocytes (CTL) for MM tumor cells were generated only when L-MM tumor hybrid (L-FM3A#2)-primed spleen cells were stimulated in vitro with a mixture of mitomycin C-treated parental FM3A/R tumor cells and L cells, but not when they were stimulated with FM3A/R or L cells alone. Killer-helper activity of L cells was replaced by a beta-interferon (IFN) preparation (L-cell IFN). Anti-(alpha+beta)-IFN antibodies completely inhibited the killer-helper activity of L-cell IFN. Several lines of evidence suggested that the killer-helper effect of L-cell IFN resulted from an action at an early step in the generation of anti-MM CTL, as follows. i) The killer-helper effect of L-cell IFN diminished as the addition was delayed during culture for 5 days. ii) The presence of L-cell IFN during the first 24hr was enough to generate a maximal CTL response. iii) Anti-(alpha+beta)-IFN antibodies showed no inhibitory activity on the generation of anti-MM CTL when they were added to the culture on day 2 or 4. It was also demonstrated that the killer-helper effect of L-cell IFN resulted from an effect on primed spleen cells, but not from an effect on stimulator cells. Furthermore, it was observed that IFN acted on nylon wool-adherent cells in the generation of anti-MM CTL. Plastic-adherent cells preincubated with L-cell IFN also enhanced the generation of anti-MM CTL when they were added to a culture where primed spleen cells were stimulated with FM3A/R tumor cells. These results suggest that activation of macrophages by L-cell IFN at the initiation step is essential for the generation of anti-MM CTL.

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Three cell lines resistant to adriamycin, melphalan and cisplatin were established in vitro from human ovarian cancer cell line A2780. Each subline showed a resistance to its inducing drug of 75-fold in the case of adriamycin, 6-fold in the case of melphalan and 11-fold in the case of cisplatin. However, all of these sublines showed collateral sensitivity to bleomycin. Approximately a 2-fold higher susceptibility to bleomycin was observed generally. The biochemical mechanisms of this collateral sensitivity are not clear at present, but the higher concentration of glutathione in these resistant tumor cell lines might be related to the high susceptibility of these resistant cells to bleomycin.

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Human recombinant interferon-γ (ReIFN-γ) and human natural interferon-γ (IFN-γ) showed significant differences in antiviral and anticellular activities. Namely, ReIFN-γ exhibited antiviral activity against Sindbis virus and anticellular activity against HeLa S₃ cells at significantly lower concentrations as compared with IFN-γ at short exposure times of between 1 and 4hr. This difference of activities was explicable in terms of different binding activities of the two IFNs to the receptors. The binding of ¹²⁵I-ReIFN-γ to the receptors was competitively decreased in a dose dependent manner by unlabeled ReIFN-γ, whereas the competition by unlabeled IFN-γ was significantly weaker. The results of pretreatment of the cells with unlabeled ReIFN-γ or IFN-γ suggested that the binding of IFN-γ to the receptors was slower than that of ReIFN-γ. ReIFN-γ and IFN-γ exhibited greater colony formation-inhibitory activity against HeLa S₃ cells, as compared with IFN-α or ReIFN-β. ReIFN-γ also inhibited DNA, RNA and protein syntheses of HeLa S₃ cells more potently than ReIFN-β. However, the 2'-5' oligoadenylate (2'-5'A) synthetase activity in ReIFN-γ-treated cells was significantly lower than that in ReIFN-β-treated cells, suggesting that 2'-5'A synthetase does not play a major role in the anticellular activity of ReIFN-γ, at least against HeLa S₃ cells.

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