We constructed a fusion plasmid, pMX-I, by which the major open reading frame, X-I, of the bovine leukemia virus (BLV) X gene was expressed under control of the mouse metallothionein promoter. pMX-I was cotransfected into CV1 monkey kidney cells together with another construct containing the BLV long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) structural gene. The result of assay of CAT synthesis suggests that the X-I product functions as a trans-acting activation factor of the BLV LTR.

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1α, 25-Dihydroxyvitamin D₃, a hormonally active form of vitamin D, induces anchorage-independent growth of BALB/3T3 A31-1-1 and NIH/3T3 cells with concomitant increase of their mRNA level of c-Ki-ras but not of c-Ha-ras or c-myc, through a receptor-mediated mechanism. Under the same conditions, 12-O-tetradecanoylphorbol-13-acetate did not induce anchorage-independent growth in these cell lines.

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The molecularly cloned, prototype human papillomavirus (HPV) 16 DNA has a single-base deletion in the El gene. We reconstructed a putative non-defective HPV 16 genome with an uninterrupted El gene, and examined its biological functions. The reconstructed HPV 16 DNA formed foci of morphologically transformed cells in transfected rat 3Y1 cell cultures (an immortalized normal cell line). The transformed rat cells contained transcriptionally active HPV 16 DNA, which appeared to be integrated within the cell DNA, and formed colonies in soft agar.

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As the analbuminemia of analbuminemic rats (NAR) is inherited as a recessive trait, the livers of F1 hybrids of Sprague-Dawley×NAR (SD× NAR) retain the capability to produce albumin. We induced albumin-negative hyperplastic nodules in the livers of NAR, isolated the nodule cells and transplanted them in the livers of SD×NAR by infusion into the mesenteric vein. When the hosts were treated with dietary 2-acetylaminofluorene plus a partial hepatectomy, numerous albumin(-) nodules of donor origin were formed within the albumin(+)livers of the hosts. This system should be useful to analyze sequential events during hepatocarcinogenesis by using a genotypic marker.

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Antitumor activity of Meth A-immune BALB/c mouse spleen cells (Meth A-Im-SPL) was assayed by a Winn test in CD-1(nu/nu) or BALB/c(nu/nu) chimeric mice which were reconstituted with the bone marrow cells of various strains of mice. It was found that Meth A-Im-SPL (L3T4⁺ T cells) neutralized the tumor in collaboration with H-2 compatible bone marrow or bone marrow-derived cells.

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Uptake and metabolism of the carcinogen 1, 2-dimethylhydrazine (DMH) were compared in isolated epithelial cells from the colon and the small intestine. A new method was developed to separate colonic epithelial cells into surface columnar cells and crypt cells without the use of any proteolytic enzymes. Colonic columnar cell-enriched fraction exhibited DMH metabolism two to three times higher than that of crypt cells. The carcinogen binding was much lower in the small intestine as compared to the colon. In the small intestine, the crypt cell-enriched fraction showed higher carcinogen binding as compared to villus cells. Pyrazole was found to inhibit DMH binding by isolated small intestinal and colonic epithelial cells. The extent of inhibition was maximum in cells showing the greatest ability to incorporate DMH.

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Intestinal metaplasia (IM) in the glandular stomach of male Wistar rats induced by oral administration of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and/or intubation of 0.1N sodium hydroxide (NaOH) was studied as follows. Experiment I, sequential study: Rats in group I were given 100μg/ml ENNG in drinking water for 12 weeks. Rats in group II were given 5ml of 0.1N NaOH by gastric intubation once a week for 12 weeks. Group III control rats were given tap water. Rats were killed from week 1 until week 69 sequentially. IM was first found at week 26 in group I and at week 58 in groups II and III, its incidence being significantly higher in group I than in the other two groups (P<0.01), but without any difference between group II and group III. Experiment II, two-stage carcinogenesis: Rats in groups I and II were treated in the same way as in experiment I, while rats in group III were given 100μg/ml ENNG for 12 weeks, followed by 0.1N NaOH once a week for 12 weeks intragastrically. All rats were killed at week 56. The numbers of metaplastic glands in groups I and III were higher than in group II. Gastric carcinomas were induced in all groups of rats treated with ENNG. The results of these two experiments show that IM is effectively induced by a carcinogen but is not enhanced by regeneration induced by alkaline treatment.

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The carcinogenic effect of estrogens, diethylstilbestrol (DES) and 17β-estradiol (E₂), and its modification by N-nitrosobutylurea (NBU) were studied in female W/Fu rats. Multiple mammary tumors (MT) of medullary carcinoma type developed at a high rate following prolonged treatment with estrogens. All MTs were located adjacent to the nipple and were slow-growing. The induction rate, multiplicity and size of estrogen-induced MTs were not influenced by pretreatment with a small amount of NBU, which alone did not induce any tumor. Ten of 12 rats (82%) receiving combined treatment with NBU and DES developed hepatic tumors (HT), while no rats in other treatment groups developed HT. All HTs were multiple nodules of various sizes bulging from the liver surface, and were considered to be neoplastic nodules. A high frequency of HT development was unexpected, because independent treatment with NBU or DES alone did not induce HT in female rats. It appears that DES played a role as a carcinogen, inducing MT and pituitary tumor (PT) through its estrogenic potency (like natural estrogen, E₂), while it also acted as a promoter or co-carcinogen in the induction of HT through its pharmacologic effects. These findings may be relevant to an increased frequency of liver neoplasm among women king oral contraceptives containing synthetic estrogens.

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The environmental occurrence and mutagenic activity of quinoline and benzoquinolines are well-documented. In this study, the relative carcinogenic activities of quinoline, benzo[f]quinoline, benzo[h]quinoline, and phenanthridine were evaluated in newborn mice. Mice were injected intraperitoneally on the first, eighth, and fifteenth day of life with 0.25, 0.5, and 1.0μmol of each of these aza-arenes. Quinoline induced a 71% incidence (P<0.005) of hepatic tumors among the male mice sacrificed at 52 weeks of age. None of the female mice treated with these aza-arenes developed hepatomas. Among the female mice treated with quinoline there was a significant development of leukemia or lymphoma (P<0.05) which was not evident among the female mice in any of the other experimental groups. Benzo[h]quinoline and phenanthridine were not carcinogenic under these assay conditions. Benzo[f]quinoline did induce an increase in the incidence of hepatomas among male mice (19% as compared to 5.9% among controls). This increase, however, was not statistically significant. These data indicate that quinoline has greater carcinogenic potential than any of these isomeric benzoquinolines in newborn mice.

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A lymphoid cell line CK-a was established from peripheral blood of an infant with acute lymphoblastic leukemia of non-T, non-B cell type with mediastinal tumor. The CK-a cells were positive for surface immunoglobulins, Epstein-Barr virus-specific nuclear antigen, HLA-DR and Leu 12 antigens, and negative for sheep erythrocyte-rosette-receptor, and Len 1, 2, 3 and 4 antigens. Budding particles were detected in electron micrographs of ultrathin sections of the CK-a cells. In the culture media of CK-a cells, particles with a buoyant density of 1.16g/ml and labeled with [³H]uridine and [³⁵S]methionine but not with [³H]thymidine were found to carry reverse transcriptase activity which preferred Mg²⁺ to Mn²⁺. Enveloped particles of 80 to 120nm in diameter were detected in the fractions at 1.16g/ml by electron microscopy. Thus, the particles had properties compatible with a definition of Retroviridae, and were tentatively named CK virus (CKV). The genome size of CKV RNA determined by agarose-acrylamide composite gel electrophoresis was 6.1±0.2kb. Immune electroblotting assay detected antibody reactive with a CKV protein with a molecular weight of 67, 000 in the serum of the patient, but not in sera of an adult T cell leukemia patient and healthy controls. No syncytia were formed by mixed cultures of CK-a and XC cells.

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Metabolic activation of phenacetin by liver microsomes proceeds via both phenetidine and N-hydroxyphenacetin to direct-acting mutagens, i.e., N-hydroxyphenetidine and p-nitrosophenetole. Five different molecular species of cytochrome P-450 have been purified from liver microsomes of drug-pretreated Wistar rats or Syrian hamsters and their abilities to activate phenetidine and phenacetin were compared using reconstituted microsome systems. High-spin forms of cytochrome P-450 purified from 3-methylcholanthrene-pretreated rats (MC-P-448-H) or hamsters (P-488 ham-II) showed higher catalytic activity for N-hydroxylation of phenetidine than three other low-spin forms of cytochrome P-450 purified from the same animals or from phenobarbital-pretreated rats. MC-P-448-H and P-488 ham-II required the presence of cytochrome b₅ for their maximum activities in the reconstituted system. The five forms of cytochrome P-450, however, exhibited no measurable activity for N-hydroxylation of phenacetin either with or without cytochrome b₅. The mutagenicity of phenacetin and phenetidine toward Salmonella typhimurium TA100 was generated when the reconstituted microsomes containing MC-P-488-H or P-488 ham-II were used as activating enzymes. From these results, it was suggested that high-spin forms of cytochrome P-450 (MC-P-448-H and P-448 ham-II) played an important role in the metabolic activation of phenacetin to the direct-acting mutagens.

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The recently increasing trend of lung cancer mortality in Japan was qualitatively analyzed. As the percentages of cases undergoing pathological autopsies in Tokyo were thirty for males and twenty-six for females during the period from 1979 to 1983, the histologically classified death rates in Tokyo could be estimated by combining the reported cases of death from lung cancer with the proportion of these of known histological type. The results indicated an increase in adenocarcinoma, and a decrease in squamous-cell carcinoma, except for the older age-group of men. The latter result suggested that the results were affected by the decreasing proportion of smokers and by the improvement in cigarette quality. The increase in small-cell and large-cell carcinoma, and the decrease in undifferentiated carcinomas, could be explained by problems associated with differing diagnostic standards. However, when these three different types of carcinoma were considered as a single type, the death rate was shown to be increased except in younger age-groups of women.

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Phenotypic markers of leukemic cells from 76 children with acute leukemia were examined. Of these cases, 7 were diagnosed as acute undifferentiated leukemia (AUL) whose leukemic cells were negative for myeloperoxidase and did not react with lineage-specific or lineage-associated monoclonal antibodies. Then, we analyzed the configuration of both immunoglobulin (Ig) and T-cell receptor β-chain (Tβ) gene in these 7 AUL cases. Three cases had no rearrangement of Ig or Tβ genes which was suggestive of non-lymphoid origin of these cases. In contrast, 4 cases showed rearrangements of Ig and/or Tβ genes. One of these 4 cases demonstrated Tβ gene rearrangement with retention of the germline configuration of Ig genes. Two cases with both Ig and Tβ gene rearrangements also showed kappa-chain gene rearrangements. These findings indicate the heterogeneity of AUL at the DNA level, and may cast new light on the early differentiation of hematopoietic progenitor cells.

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The role of L3T4-positive lymphocytes in the generation of anti-tumor immunity against syngeneic murine plasmacytoma was investigated. The induction of in vitro primary and secondary cytotoxic T lymphocytes against MOPC-104E plasmacytoma of BALB/c origin was completely inhibited by the addition of monoclonal anti-L3T4 antibody (GK1.5) during the culture period. The possible participation of L3T4-positive lymphocytes in the induction of cytotoxic T lymphocytes was confirmed by the pretreatment of non-immune or immune splenocytes with GK 1.5 plus complement followed by in vitro stimulation with the tumor. he treatment abrogated the ability to generate killer T cell activity. Because the effector killer T lymphocytes were not affected by GK1.5 plus complement treatment, the results strongly suggest that the L3T4-positive lymphocytes might be acting as helper cells for the generation of killer T lymphocytes. The in vivo effect of GK1.5 on anti-tumor immunity was then investigated. Tumor cell growth in mice which received GK1.5 injections was significantly accelerated and the mean survival time of the mice was shorter than that in control groups. On the other hand, mice with established anti-tumor immunity produced by hyperimmunization of tumor cells were affected differently from non-immune mice, showing 50% tumor incidence on day 30 after viable tumor challenge. The present study supports the contention that the L3T4-positive T lymphocytes, probably helper T cells, play a crucial role in the establishment of anti-tumor immunity.

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Three monoclonal antibodies (MAbs) of different immunoglobulin subclasses against MH134 murine ascitic hepatoma cells, detecting the same antigenic determinant of tumor-associated antigen of the tumor cells, were tested for their ability to produce a synergistic antitumor effect with Mycobacterium bovis BCG in C3H/HeN mice. 12A2 (IgG2a) induced both antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against the tumor cells. C3H/HeN mice were inoculated ip with MH134 tumor cells (day 0), and received an ip injection of BCG (day 1) and/or 12A2 (day 5). The combined therapy with BCG and 12A2 brought about a significant prolongation of the survival period, whereas either BCG or the MAb alone exhibited poor therapeutic effectiveness. 11G2 (IgG1), inducing ADCC but not CDC against MH134 tumor cells, was shown to exhibit antitumor effects as potent as those of 12A2, when used in combination with BCG. However, 7C2 (IgM), capable of inducing CDC but not ADCC to the tumor cells, produced no apparent synergistic effect with BCG. ADCC of BCG-induced peritoneal cells was mediated by the adherent cell population of the cells and abolished by the addition of carrageenan, suggesting that the effector cells of the cytotoxicity were macrophages. Moreover, carrageenan abolished the combined antitumor effect of BCG and these MAbs in the serological Winn assay. These results suggest that activated macrophages play a major role in the synergistic antitumor effect of BCG and MAbs capable of inducing ADCC against MH134 tumor.

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Purified human natural tumor necrosis factor (n-TNF) was prepared by stimulating human leukemic B cell line (BALL-1) with Sendai virus. The colony formations of all of 18 human cancer-derived abnormal cell lines were suppressed by 10¹-10⁶U/ml of n-TNF, while n-TNF was nontoxic to all human normal fibroblast cells. This in vitro inhibition of cell growth was reversible. In breast adenocarcinoma MCF7 cells treated with n-TNF a specific decrease of DNA synthesis was observed, and DNA histograms showed a block at G₁ in the cell cycle. In vivo studies revealed that n-TNF suppressed the tumor growth of murine Meth A sarcoma, human renal adenocarcinoma (ACHN), malignant melanoma (SK-MEL-28) and glioblastoma (U-373 MG). Isobologram analysis showed that n-TNF synergistically inhibited cell growth in combination with human natural interferon (IFN)-α. In vivo synergism of n-TNF and IFN-α was also found in the U-373MG tumor model implanted into nude mice.

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