Human squamous cell carcinoma tissue was screened to determine the epidermal growth factor (EGF) receptor level. In 9 out of 15 cases, increased EGF binding was observed relative to normal adjacent tissue. In two tissue samples (SCE1 and SCL1), amplification of the EGF receptor gene and increased mRNA level were also observed. In two other cases (SCE2 and SCL3), EGF receptor gene amplification did not occur. We propose that there is an alternative mechanism for EGF receptor hyperproduction, independent of gene content, active in these tissues. A possible role of EGF receptor hyperproduction is envisioned in human neoplasia.

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We have detected by DNA hybridization human papillomavirus (HPV) types 16 and 18 DNA sequences in 19 and 3, respectively, out of 56 cervical carcinomas from Japan. Eighteen out of 19 HPV 16-positive specimens were from squamous cell carcinomas, whereas the three HPV 18-positive specimens were from a squamous cell carcinoma (1/50), an adenosquamous carcinoma (1/3), and an adenocarcinoma (1/3). The occurrence of HPV 16 DNA decreased in patients over 60 years old (≤60 years, 15/34 (44%); 60 years<, 4/22 (18%)) (P<0.05).

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During the course of screening of agents active in converting the transformed morphology of Rous sarcoma virus-infected rat kidney cells to the normal morphology, we identified an active substance produced by Streptomyces sp. MH237-CF8 as herbimycin. Herbimycin converted almost all cells into the normal morphology. The antibiotic was found to be an inhibitor of p60^src-associated protein kinase in the cells.

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The mechanism of growth enhancement of 7, 12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors by high fat diet was investigated in 169 female Sprague-Dawley rats fed a semisynthetic diet containing 0, 8 or 40% corn oil by weight. The animals, most of which were intubated with 10mg of DMBA twice during the 8th week of age, were fed an unsupplemented or fat-supplemented diet for 2 months from the 12th to the 21st week of age and finally killed during the 21st week of age. Compared to the rats fed a fat-free diet, a significantly increased yield of mammary adenocarcinoma with a shorter latency was observed in the rats fed a 40% corn oil diet. DNA synthesis of the biopsied mammary tumors more than 1 month after diet switch-over was estimated in terms of the rate of [³H]thymidine incorporation into the tissue. Significantly higher DNA synthesis of the mammary tumors biopsied from proestrous hosts was found, as compared with the tumors in similar hosts at other stages of the estrous cycle. The greatest enhancement of DNA synthesis in small and rapidly growing tumors was observed in the 40% corn oil diet group. Feeding with 40% corn oil diet always resulted in elevation of the weight percentage of linoleic acid in both simple lipids and phospholipids of the mammary tumors. These data indicate that the effects of dietary fat on mammary tumor growth might be mediated by the enhancement of tumor response to certain hormones, and that the enhanced responsiveness of tumors was associated with increased linoleic acid in tumor lipids.

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7-Hydroxymethyl-12-methylbenz[a]anthracene (7-HMBA) and 12-hydroxymethyl-7-methylbenz[a]anthracene (12-HMBA), carcinogenic major metabolites of 7, 12-dimethylbenz[a]anthracene (DMBA) in untreated rat liver, showed high mutagenic activities toward Salmonella typhimurium TA 98 after preincubation with a sulfotransferase-PAPS system consisting of ATP, sodium sulfate, and a post-mitochondrial fraction (S-9) or a soluble supernatant fraction (S-105) from untreated rat liver. The 7- and 12-HMBAs themselves induced His⁺ mutation in TA 98 only slightly after preincubation with S-9 in the presence of an NADPH-generating system. Mutagenicity of DMBA toward TA 98 after preincubation with S-9 in the presence of the NADPH-generating system was remarkably enhanced by the addition of ATP and sodium sulfate. The active metabolites, 7-HMBA sulfate and 12-HMBA sulfate, were isolated from these preincubation systems and identified by comparison with the corresponding synthetic specimens. The sulfuric acid ester conjugates were potent mutagens toward TA 98 in the absence of rat liver subcellular fractions. The conjugates bound covalently at significant rates to calf thymus DNA as well as to S-105 proteins at 37° and pH 7.4 through the 7- or 12-methylene carbon with concomitant loss of their sulfate group. In the presence of S-105, glutathione inhibited the mutagenicity of the metabolically formed or exogenously added 7- and 12-HMBA sulfates. The non-mutagenic glutathione conjugates were isolated from the incubation mixtures and identified as S-(12-methylbenz[a]anthracen-7-yl)methylglutathione from 7-HMBA or its sulfate and S-(7-methylbenz[a]anthracen-12-yl)methylglutathione from 12-HMBA or its sulfate.

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The protective effect of progesterone or tamoxifen, an antiestrogenic agent, was investigated in estrogen-induced mammary carcinogenesis. Multiple mammary tumors (MT) of tubular or medullary carcinoma type developed at a high rate following prolonged treatment of ovariectomized W/Fu rats with diethylstilbestrol or 17β-estradiol. All MTs were located adjacent to the nipple and were slow-growing. The induction rate, multiplicity and size of estrogen-induced MTs were reduced by the simultaneous administration of either progesterone or tamoxifen. The estrogen-induced pituitary tumorigenesis was effectively inhibited by tamoxifen treatment, but it was not affected by progesterone. The results indicated that the inhibitory effect of progesterone or tamoxifen in estrogen-induced carcinogenesis is attributable to interference with the binding of estrogen to the estrogen receptors on the target cells.

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A simultaneous case-control study on stomach cancer and colo-rectal cancer involving 93 cases with stomach cancer, 93 cases with colo-rectal cancer and 186 controls was conducted using a common questionnaire at the Aichi Cancer Center Hospital in 1981-83. A fondness for salty tastes, especially salted foods such as pickled hakusai (vegetable) and dried & salted fishes, which are typical traditional Japanese foods showed a significantly positive association with stomach cancer (relative risk(RR)=2.60, P<0.01). On the other hand, the habit of eating a western-style breakfast, particularly for 10 years or more made a significant contribution to the risk of colon cancer (RR=2.24, P<0.05) but conversely decreased the risk of stomach cancer (RR=0.50, not significant (NS)) and rectal cancer (RR=0.40, NS). In this study, relatively frequent intakes (4 times/week) of some vegetables, i.e. pumpkin, green pepper, onion and cabbage, showed high relative risks for both stomach and colon cancers, contrary to the findings of previous epidemiological studies. Cigarette smoking increased the risk of stomach cancer (RR=1.99, NS) but decreased that of colon cancer (RR=0.61, NS). There was no positive relation between drinking and cancer at any site. Some other factors with opposite effects on the two contrasting cancers and some independent factors were identified in this comparative casecontrol study.

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In order to study the effect of urbanization on the epidemiological features of colo-rectal cancer in Japan, the age-adjusted death rates (AADRs) for right-sided (cecum, ascending and transverse) colon, left-sided (descending and sigmoid) colon and rectum cancers during three periods, 1969-71, 1974-76 and 1979-81, were compared among five areas with different population sizes; ten metropolitan cities (>1, 000, 000), large cities (1, 000, 000-150, 000), medium-sized cities (150, 000-50, 000), small cities (<50, 000) and counties. The AADR for left-sided colon cancer has been increasing in recent years, especially in males, and was higher in urban areas than in rural areas, which may imply that the recent increase in the incidence of left-sided colon cancer is associated with an urbanization of the life styles of Japanese people. In Japan, the daily consumption of carbohydrate has been decreasing, but that of total fat has been increasing in recent years, and the ratio of fat/carbohydrate consumption as an energy source was strongly correlated with the AADRs for left-sided colon cancer among the five areas in the three periods. From this correlation analysis, it was suggested that urbanization of life style, namely, the recent westernization of eating habits of Japanese people might be associated with the recent increase of the AADRs for colon cancer, especially of left-sided colon cancer, in Japan.

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Synergistic cell killing of human lymphoblastic leukemia cell line, CCRF-CEM, occurred when hydroxyurea (HU) was administered before 1-β-D-arabinofuranosylcytosine (ara-C). At the optimal dose of HU (1mM), the ara-CTP concentration increased 4-fold and the intracellular accumulation of ara-C increased 4-fold, while the dCTP concentration decreased by more than 50%. Increased intracellular accumulation of ara-C after HU treatment was also observed in human acute myelogenous leukemic cells in circulating blood. Therefore, the synergistic cell kill of HU and ara-C may be the consequence of greater inhibition of DNA polymerase by the increased level of ara-CTP in the presence of the decreased concentration of the natural substrate of this enzyme, dCTP. This synergism was not due to an increased incorporation of ara-C into DNA since the treatment of cells with HU did not enhance the ara-C incorporation into DNA but rather suppressed it.

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Studies were conducted to characterize the nuclear RNA (nRNA) species that were present in rat liver but absent in the hepatoma. Nuclear RNA was compared between Donryu rat liver and AH136B hepatoma, an azo dye-induced transplantable cell line, by DNA-RNA competitive hybridization. The hepatoma lacked 13-14% of nRNA according to measurements of radioactivity of the hybridized ³²P-labeled liver nRNA, and this loss was shown to be due to the failure to transcribe such RNA rather than to the deletion of the relevant DNA in the genome. Characterization of the lost RNA was first attempted by fractionating liver nRNA by density gradient sedimentation and polyacrylamide gel electrophoresis. A comparison of the additive effects of the fractionated RNA's in the competitive hybridization indicated that the pertinent RNA was present in the large RNA molecules (>14S), not in the low molecular weight RNA's. Then poly(A) nRNA was found to show a strong additive effect in the competitive hybridization while nucleolar RNA showed little additive effect, indicating that the pertinent RNA was present in the heterogeneous nRNA, not in the ribosomal precursor. Further characterization was done by fractionating DNA with regard to the repetition in the genome. A comparison of the competitive hybridizations on the fractionated DNA's showed that the loss occurred mostly in RNA transcribed from highly repetitive DNA. In conclusion, the RNA species lost in the hepatoma were components of heterogeneous nRNA transcribed from highly repetitive DNA.

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At 22hr after ip injection into male Wistar rats and BALB/c mice, chlorobenzene was covalently bound to DNA, RNA and proteins of the liver, kidney and lung, as has been found with various weak carcinogens. A microsome-mediated interaction with DNA occurred in vitro. The interaction was enhanced by pretreatment in vivo with phenobarbitone but was suppressed by addition of 2-diethylaminoethyl-2, 2-diphenylvalerate•HCl in vitro. These results indicate the involvement of cytochrome P-450. Liver microsomes were efficient bioactivators, whereas cytosol was ineffective. The extent of in vitro interaction of chlorobenzene with synthetic polyribonucleotides was of the same order as that with DNA. Finally, ultraviolet irradiation (λ=254nm or λ_max=365nm) activated this environmental contaminant to forms capable of interacting with DNA. The results represent evidence for genotoxicity of chlorobenzene.

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Human chorionic gonadotropin (hCG) highly purified from the urine of patients with trophoblastic diseases (choriocarcinoma and hydatidiform mole) and from healthy pregnant women contains four asparagine-linked sugar chains in one molecule. Comparative studies of the sugar chains released by hydrazinolysis revealed that the structures of the sugar chains of choriocarcinoma hCGs are quite different from those of mole and normal hCGs. The carbohydrate structures of mole hCGs are the same as those of normal hCG, while all choriocarcinoma hCGs examined contain two triantennary, two unusual biantennary, and one monoantennary complex-type sugar chains which are not found in normal hCGs. The triantennary and unusual biantennary sugar chains contain the ±NeuAcα2→Galβ1→4GlcNAcβ1→ 4Manα1→3 group in common. The total amounts of fucosylated sugar chains in choriocarcinoma hCGs are twice those found in the hCGs of other groups. Therefore, the appearance of the N-acetylglucosaminyl transferase responsible for the formation of the GlcNAcβ1→4Manα1→3 group and the increase of the fucosyl transferase responsible for the formation of the Fucα1→6 GlcNAc→Asn group appear to be specific characteristics of malignant transformation of the trophoblast, and the structural changes of sugar chains could serve as useful markers for the diagnosis of choriocarcinoma.

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The aim of this work was to selectively stimulate N-glycosylation of proteins in cultured AH 70Btc hepatoma cells. Dolichyl phosphate, α-dihydrodecaprenyl phosphate and solanesyl phosphate were entrapped in egg lecithin liposomes and supplied to the cells. After treatment with 0.6-15nmol of the polyprenyl phosphates/3ml/dish for 48hr, the incorporations of [¹⁴C]glucosamine into N-linked saccharide chains of glycoproteins and into lipid intermediates were increased to various extents. The stimulation by α-dihydrodecaprenyl phosphate was most significant and 15nmol of the lipid/dish increased the incorporation into N-linked saccharide chains of glycoproteins by about 80%. The incorporations of [¹⁴C]glucosamine into O-linked saccharide chains and of [¹⁴C]leucine into proteins were not changed significantly by α-dihydrodecaprenyl phosphate. When the N-linked glycopeptides prepared from the cells and those from the cells treated with the polyprenyl phosphate were compared by Sephadex G-50 column chromatography, no significant difference was observed. Analysis of reduced cellular glycoproteins by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis showed that α-dihydrodecaprenyl phosphate increased the incorporation of [¹⁴C]glucosamine into N-linked saccharide chains of certain high-molecular-weight glycoproteins. In addition, the polyprenyl phosphate enhanced the adhesiveness of the cells to the substratum, probably as a result of the stimulation of N-glycosylation of proteins.

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MT6 tumor is a transplantable ovary- and prolactin-independent rat mammary carcinoma containing estrogen and progesterone receptors. The effects of daily injections of 1mg or 10μg of 17β-estradiol for 28 days on the morphological changes and the proliferation and death of MT6 cells grown in male inbred Sprague-Dawley rats were investigated. Uptake values of 5-[¹²⁵I]iodo-2'-deoxyuridine per g of living tumor, used as an index of cell proliferation, were not significantly reduced by treatment with 1mg of 17β-estradiol. When tumor cell proliferation was evaluated by labeling with 5-[¹²⁵I]iodo-2'-deoxyuridine just before the treatment, the radioactivity incorporated by the whole tumor remained almost unchanged in intact rats but was decreased slightly on day 21 and significantly on day 28 of the treatment in the rats treated with 1mg of 17β-estradiol. Secretory changes characterized by the appearance of secretion in the lumina, vacuolation in the cytoplasm, marked increase in vesicular endoplasmic reticulum, accumulation of mature protein secretory granules and abundance of lipid droplets were observed in MT6 cells in rats treated with 1mg of 17β-estradiol from the 14th day of treatment. On the 28th day, necrosis of MT6 cells with secretory changes was found. These changes were not found in intact rats or rats treated daily with 10μg of 17β-estradiol. These findings suggest that the inhibitory effect of daily injections of 1mg of 17β-estradiol on the growth of ovary- and prolactin-independent MT6 tumor is mainly due to secretory changes followed by loss of tumor cells induced by pharmacological doses of estrogens.

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Proliferative activity of normal and carcinogen-altered hepatocytic populations was investigated in Wistar rats of various ages ranging from 5 to 109 weeks old before and after a two-thirds hepatectomy. γ-Glutamyltranspeptidase(GGT)-positive hepatocytic foci were induced by administration of a single dose of diethylnitrosamine. The incidence of nuclear labeling with [³H]thymidine was examined using simultaneous GGT histochemistry and radioautography. The [³H]thymidine index of hepatocytes in the GGT-positive foci was not greater than that of normal hepatocytes in both young and old non-hepatectomized rats. GGT-positive hepatocytes responded by proliferating after a two-thirds hepatectomy in all age groups. Although the response of normal hepatocytes to the growth stimulus markedly decreased in old rats, GGT-positive hepatocytic populations retained relatively high responses to the growth stimulus. These observations indicate that in old rats carcinogen-altered hepatocytes have a greater growth advantage than normal hepatocytes to exogenous and endogenous proliferative stimuli.

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