Daily administration of purified human granulocyte colony-stimulating factor produced by E. coli (Hu-G-CSF) accelerated the recovery from neutropenia induced by total-body irradiation in mice. Granulocyte-macrophage progenitors assayed as colony-forming unit granulocyte/macrophage (CFU-GM) in spleen were markedly increased in number by Hu-G-CSF, and the accelerated recovery from neutropenia may be due to the stimulation of granulocytopoiesis by the G-CSF from progenitor cells.

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Glycyrrhizin sulfate (GLS) was synthesized and investigated for antiviral effect on the human immunodeficiency virus (HIV) in vitro in comparison with the parental anti-HIV compound glycyrrhizin (GL). In MT-4 cells after HIV infection, the virus-induced cytopathic effect and the expression of viral antigens were inhibited by 0.25mg/ml (0.184 mM) of GLS. Moreover, GLS completely inhibited HIV-induced plaque formation in MT-4 cells at a concentration of 1mg/ml (736μM), the 50% inhibitory dose being 0.055mg/ml (40μM). GLS was found to be an efficient inhibitor of reverse transcriptase. The effect of GLS was 4 times stronger than that of GL in molar terms.

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We have regionally localized the two human tyrosine kinase proto-oncogenes, c-ros-1 and flt, by in situ hybridization. The c-ros-1 has been mapped to 6q22 and the flt to 13q12.

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We isolated several mouse erythroleukemia (MEL) cell lines, deficient in in vitro erythroid differentiation, and examined the change in level of c-myc mRNA after exposing the cells to dimethyl sulfoxide (DMSO). The results showed all the mutant MEL cells to exhibit a similar change in c-myc mRNA level to that in parental (DS19) cells with regard to both an initial decline and a subsequent increase to the pretreatment level. The c-myc mRNA level did not, however, decline again thereafter, as observed in the parental cells, but remained at the pretreatment level, even on prolonged culture in the presence of DMSO. Essentially the same results were obtained for the parental cells in the presence of DMSO and either 12-O-tetradecanoylphorbol 13-acetate (TPA) or dexamehasone, specific inhibitors of MEL cell differentiation. These results suggest that the crucial stage in MEL cell differentiation, regarding c-myc mRNA level, is the late stage, when the MEL cells become committed to differentiation.

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Sex chromosome abnormalities in human stomach and lung cancers from 33 male patients were examined by Southern blot hybridization with pDP34 DNA probe, which recognizes X and Y chromosome-linked restriction fragment length polymorphisms (RFLPs). Contrary to the recent cytogenetic observations showing high incidence of loss of the Y chromosome in solid tumors, loss of the Y chromosome was observed in only 3 of 21 stomach cancers and 2 of 12 lung cancers. Gain of the Y chromosome was found in one of 12 lung cancers, but not in any of the stomach cancers. No X chromosome abnormality was found in any of these 33 stomach cancers and lung cancers.

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The distributions of gamma-glutamyltransferase (GGT) and the placental form of glutathione S-transferase (GSTP) were examined in the livers of 18-month-old and 23-month-old SPF F344/DuCrj rats of both sexes. The number of the enzyme-altered foci in males was greater than in females, and it increased with age in animals of both sexes. Histologically, most of the foci in males consisted of eosinophilic or clear hepatocytes, while those in females were predominantly basophilic. More than 75% of the foci in males stained positively for GGT and GSTP. In contrast, more than 80% of the foci in females were GGT- and GSTP-negative. Thus, though both enzymes have been widely used for analysis of carcinogen-induced hepatocarcinogenesis, it appears that GGT and GSTP are inappropriate as markers of preneoplastic lesions in natural hepatocarcinogenesis in female rats.

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The mechanism of the anti-promoting effect of the prostaglandin (PG) synthesis inhibitor indomethacin in colon carcinogenesis was investigated. Male Sprague-Dawley rats received 0.002% water solution of indomethacin as drinking water freely for 3 days, then a subcutaneous injection of various doses of PGE₂ and/or an intrarectal instillation of 12μmol of sodium deoxycholate as a colon tumor promoter. Ornithine decarboxylase (ODC), a marker of tumor promotion, in the distal colonic mucosa was assayed at 4hr after deoxycholate instillation. Indomethacin significantly suppressed the deoxycholate-augmented increase of ODC activity, while exogenous PGE₂ restored or further increased the augmented ODC activity. The amount of PGE₂ and the level of ODC activity were well correlated. However, PGE₂ alone without deoxycholate did not increase the activity. Deoxycholate markedly increased colonic mucosal PGE₂ at 1hr after the instillation, and indomethacin decreased it. The results indicate that PGE₂, the production of which is stimulated in the colonic mucosa by deoxycholate, is involved in the induction of colonic mucosal ODC. This is probably why PG synthesis inhibitors may inhibit the tumor promotion and prevent cancer development in the colon.

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Modulation of thymic microenvironments during ontogeny and lymphomagenesis in mice was studied with two rat monoclonal antibodies (moAb) which recognized distinct subpopulations of thymic epithelial reticular cells (TER). In adult thymus, the TER subpopulation stained by moAb B6TS-1 was localized in the subcapsular zone, cortico-medullary junction, and medulla. In fetal thymus, it was initially distributed throughout the rudiment, but after day 16 of gestation, it was rapidly redistributed to the locations seen in adult thymus. From an early stage of thymic lymphomagenesis, the TER bearing mB6TS-1 (epitope defined by moAb B6TS-1) in the corticomedullary junction, in particular those associating with small blood vessels, proliferated and formed a characteristic network throughout the thymus, in which numerous growing lymphoma cells were entrapped. On the other hand, moAb AKTS-1 stained another TER subpopulation that was localized in the cortex in both fetal and adult thymus. Unlike mB6TS-1⁺ TER, mAKTS-1⁺ TER became increasingly sparser during lymphomagenesis. Selective proliferation of the mB6TS-1⁺ TER subpopulation in the cortico-medullary junction was seen in spontaneous, radiation-induced, and chemical-induced mouse thymic lymphomas. The possible biological significance of such modulation of thymic microenvironments in the natural history of lymphomagenesis is discussed.

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The genotoxicities of 8 nitroarenes, i.e., 1-nitropyrene, 1, 3-dinitropyrene, 1, 6-dinitropyrene, 1, 8-dinitropyrene, 2, 7-dinitrofluorene, 3-nitrofluoranthene, 1-nitro-3-hydroxypyrene and 1-nitro-3-acetoxypyrene, were examined in DNA repair tests using human isolated hepatocytes. Out of the tested nitroarenes, 5 compounds, i.e., 1-nitropyrene, 1, 3-dinitropyrene, 1, 6-dinitropyrene, 1, 8-dinitropyrene and 1-nitro-3-acetoxypyrene, clearly elicited positive responses of DNA repair. Among the chemicals which elicited positive responses, the levels of unscheduled DNA synthesis induced by the three dinitropyrene isomers were much higher than those of the other nitroarenes. Three chemicals, i.e., 2, 7-dinitrofluorene, 3-nitrofluoranthene and 1-nitro-3-hydroxypyrene, elicited negative responses. The negative responses of 2, 7-dinitrofluorene and 3-nitrofluoranthene, which had been positive in DNA repair tests with rodent hepatocytes, suggest some species differences between humans and rats in the metabolic activity of hepatocytes toward these agents.

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Tests were performed to study the transforming growth factor (TGF) activity in samples of pleural fluid and ascites in patients with solid tumors and non-neoplastic diseases such as congestive heart failure and liver cirrhosis. The measurement of the activity was carried out in terms of colony formation in soft agar with NRK cells as indicator cells. When the fluid samples were directly assayed, 24% (4/17) of the cancer cases and 1 of the 4 control cases were positive. The eluate of Bio-Gel P-60 gel filtration was positive in all cases. β-Type as well as α-type TGF activity was found in pleural and peritoneal effusions not only from cancer patients, but also from patients with non-malignant diseases.

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The effect of quercetin, a flavonoid, on the in vitro transformation of BALB 3T3 cells was examined. Quercetin at a low concentration (5μg/ml) markedly suppressed the promoting action of 12-O-tetradecanoyl phorbol-13-acetate (TPA, 10μg/ml) in this transformation system using 20-methylcholanthrene (MCA, 1μg/ml) as the initiator. At a concentration higher than 5μg/ml, however, quercetin enhanced transformation by MCA. Quercetin itself was found to induce direct transformation weakly, but definitely, at a slightly higher concentration than that for enhancing MCA-induced transformation. These results indicate that quercetin has pleiotropic effects on the transformation of BALB 3T3 cells.

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Human colorectal normal epithelia and tubular adenomas were studied by DNA-cytofluorometry using free cell nuclei isolated from formalin-fixed and paraffin-embedded specimens. All the normal epithelia (30 cases) showed low proliferative activity, no polyploid cells and no aneuploid cell clones at any age in either sex. The mean diploid G₁/G₀ fraction (DF) of the normal epithelia was 95.9%. The adenomas, on the other hand, showed increased proliferative activity usually in accordance with the grade of atypia. The mean DF values of the adenomas with mild atypia (31 lesions) and moderate atypia (21 lesions) were 89.4% and 85.6%, respectively. The adenomas, irrespective of their grade of atypia, occasionally showed low proliferative activity, while in the adenomas adjacent to a carcinoma, the proliferative activity was relatively high even though their atypia was mild. Neither polyploid cells nor aneuploid cell clones were found in the adenomas. Therefore, polyploid and aneuploid cell populations are characteristics of carcinoma of the colon and rectum, and this criterion should be useful in investigations of the genesis of carcinoma in the human colorectal mucosa.

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Human chorionic gonadotropin (hCG) from urine of patients with trophoblastic diseases was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blotting using specific antibodies. Western blotting using anti-hCGβ carboxy-terminal peptide (CTP) revealed that the molecular weights of the β subunits of the three molar hCG samples were identical to that of standard hCGβ, but those of choriocarcinoma hCG samples were individually different. In the five choriocarcinoma hCG samples, the β subunits of three samples were apparently larger than standard hCGβ, while those of two samples were smaller than standard hCGβ but larger than desialylated standard hCGβ. These results suggest that structural changes of carbohydrates of hCG, which may be induced by malignant transformation of the trophoblast, can be observed as differences in molecular weight. In SDS-PAGE under nonreducing conditions, Western blotting using anti-hCGβ-CTP revealed a single band corresponding to hCGβ. Under reducing conditions with dithiothreitol (DTT), however, a second low-molecular-weight material (called here CTP') could be observed with anti-hCGβ-CTP. All five choriocarcinoma hCG samples were much more susceptible than standard hCG to cleavage of CTP' by DTT. We conclude that estimations of molecular weight and susceptibility to DTT reduction of urinary hCG by Western blotting using specific antibodies are useful in the diagnosis of choriocarcinoma.

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Male ACI/N rats were treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in the drinking water, and in conjunction with histological examination, the changes of the expressed cytochrome P-450 components in the urothelium and other tissues (liver, kidney, esophagus, intestines) were examined by means of immunohistochemistry. Frozen tissue sections were prepared and immunostained with anti-rat cytochrome P-450 monoclonal antibodies and an avidin-biotin-peroxidase complex. Monoclonal antibodies used were APH-3 and APH-8 raised against a high-spin form of cytochrome P-448, APL-1 and APL-2 against a low-spin form of cytochrome P-448, and APF-3 against cytochrome P-450. BBN-induced qualitative and quantitative changes of cytochrome P-450 components recognized by these monoclonal antibodies were not observed in tissues other than the bladder. Untreated rat bladder epithelium was not stained with any of these 5 monoclonal antibodies. The treatment with BBN for more than 3 weeks, however, resulted in the expression of cytochrome P-450 component(s) recognized by APH-8 antibody. This cytochrome P-450 component increased with the advance of carcinogenic changes in the urothelium. The component reactive with AHP-8 was also detected in the cancer tissues of transplantation lines of rat bladder cancers. In contrast, the cytochrome P-450 components recognized by APL-1, APL-2 or APF-3 were undetectable or present at low levels throughout the BBN carcinogenesis. These results suggest that a certain cytochrome P-450 component(s), probably a high-spin form of cytochrome P-448, is selectively induced in urothelium in association with neoplastic bladder lesion.

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The intraportal implantation of mouse transplantable colon adenocarcinoma 26 was investigated as an experimental liver metastasis model of colorectal cancer. CDF₁ male mice (5 weeks old) were laparotomized under pentobarbital sodium anesthesia, and colon 26 tumor cells (1×10, 1× 10³, or 1×10⁵ cells) were inoculated into the superior mesenteric vein. On day 21 after inoculation, mice inoculated with 1×10³ tumor cells showed five to 12 colonies of liver metastasis (mean, 9.2 colonies; n=6). The survival time was 27 to 36 days (median, 27 days; n=5) in these mice. When mitomycin C (MMC) was administered into the superior mesenteric vein 15min after the inoculation of tumor cells, the number of metastatic foci in the liver was strongly inhibited; 81.1% and 100% inhibitions were observed in the groups given 4mg/kg and 6mg/kg of MMC, respectively. The mouse colon 26 model seems to be one of the more useful experimental models for evaluating treatments for the prevention of liver metastases from colorectal cancer.

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The accumulation of cytosine arabinoside-5'-triphosphate (ara-CTP), the activities of nucleotide-metabolizing enzymes and the nucleoside transport capacity were examined in eleven human leukemic cell lines differing in phenotype. The highest amount of ara-CTP was accumulated in T-acute lymphoblastic leukemia cells (T-ALL), followed by myeloid leukemia cell lines (AML), and the least accumulation was observed in common acute lymphoblastic leukemia (c-ALL) and B-acute lymphoblastic leukemia cells (B-ALL). The levels of enzymes involved in ara-C metabolism (deoxycytidine kinase, pyrimidine monophosphate kinase and deoxycytidylate deaminase) did not correlate with ara-CTP accumulation. The sensitivity of the leukemic cell lines to ara-C, which was measured in terms of decrease of clonogenic survival, correlated with the amount of ara-CTP formed. Moreover, the nucleoside transport capacity, estimated from the binding of the radiolabeled nucleoside analogue, [³H]nitrobenzylthioinosine, showed a good correlation with ara-CTP accumulation. The mean numbers of nucleoside-binding sites in T-ALL cells were significantly greater than in B-ALL cells. We conclude that the cellular nucleoside transport capacity is the most important factor for the formation and accumulation of ara-CTP in the cells.

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The drug sensitivity of retinoblastoma cells obtained from 14 fresh primary materials (13 from enucleation and 1 from autopsy) and 2 cultured lines (Y-79 and WERI-Rb1) was determined using the human tumor clonogenic assay developed by Hamburger and Salmon. Components of the conventional soft agar medium were slightly modified to make them suitable for growing primary retinoblastoma cells. More than 5 colonies were formed by all 14 primary samples tested from the 500×10³cells plated. More than 30 colonies per dish were formed from the 13 samples, with a median plating efficiency of 0.033% (0.005-0.400), and these were used in the in vitro measurements of drug chemosensitivities. They showed homogeneous sensitivity to the representative alkylating agent L-phenylalanine mustard; 13 out of 14 showed a decrease in the colony formation of more than 70%. The other drugs which were effective (more than 70% colony inhibition) against the primary retinoblastoma cells were: doxorubicin (7 out of 13), mitomycin C (7 out of 13), actinomycin D (4 out of 13), cis-diamminedichloroplatinum(II) (3 out of 13), nimustine (1 out of 13), and peplomycin (1 out of 13). Vincristine, bleomycin, 5-fluorouracil, methotrexate, dacarbazine, and cytosine arabinoside were not effective. When the chemosensitivity of retinoblastoma cells of the two established cell lines was examined by the same method, only L-phenylalanine mustard was effective against Y-79, and no drug was effective against WERI-Rb1.

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