A single topical application of 7, 12-dimethylbenz[a]anthracene (DMBA) followed by twice-weekly 12-O-tetradecanoyl phorbol-l3-acetate (TPA) treatment was given to the skin of female CD-1 mice, and soft agar colony formation of the epidermal cells was evaluated periodically during the treatment. From the second week, colony growth was observed and the number of colonies increased with time, reaching a significant level from the fourth week. Colony formation was observed earlier than papilloma development in the mouse skin. Soft agar colony-forming assay might be a useful tool to analyze the early events in carcinogenesis.

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The effects of minute amounts of cigarette smoke with or without nebulized N-nitroso-N-methylurethane (NMUT) on the respiratory epithelia of mice were examined. The first group received the combination of NMUT nebulization and cigarette smoke inhalation, the second group NMUT nebulization only, the third group cigarette smoke inhalation only and the fourth group no treatment. The first group showed squamous cell carcinomas in the nasal cavity (4 out of 10mice), trachea (4 out of 10 mice) and lung (2 out of 10mice). In the second group squamous cell carcinomas with keratosis were found in the nasal cavity (2 out of 10mice) and in the lung (1 out of 10 mice). Furthermore, basal cell hyperplasias, squamous metaplasias and dysplastic changes were found in these two groups. The third group showed basal cell hyperplasias and squamous metaplasias in the nasal cavity and hypertrophy of Clara cells. No marked changes of epithelia of the respiratory tract in the fourth group were seen. Adenomas and adenocarcinomas were not found in any group. Statistically, there is a significant difference in the incidence of dysplastic change of the trachea between group 1 and group 2 (P<0.05). These results suggest that minute amounts of cigarette smoke, in combination with carcinogens, may have cocarcinogenic action and that squamous cell carcinoma may pass through the course of basal cell hyperplasia, squamous metaplasia and dysplastic change of the epithelia.

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Highly lymphomagenic mouse type-C viruses were generated from radiation- or chemically-induced T-cell lymphoma cell lines of NFS/N mouse origin infected with a non-oncogenic ecotropic virus E4. By analysis of these progeny viruses, the following results were obtained. 1) The viruses were lymphomagenic in neonatally inoculated NFS/N and C3H/He mice and W/Fu rats but not in Balb/c and C57BL/6N mice, indicating that they possess the Fv-1ⁿ tropism of exogenously infected parent virus. 2) Lymphomagenic viruses consisted of plural viral subpopulations. Recombinant mink cell focus-inducing (MCF) and ecotropic viruses were cloned from them. Inoculation of either MCF or ecotropic virus alone or both viruses together did not cause lymphoma in NFS/N mice and there was no evidence of viral replication in the recipients. 3) Inoculation of either MCF- or ecotropic virus-infected NFS-ME cells alone did not cause lymphoma development in pre-irradiated NFS/N mice, while transplantation of both MCF- and ecotropic virus-infected NFS-ME cells resulted in the development of lymphomas of host origin. These results show that lymphomagenic MCF virus was generated through the recombination of E4 viral genome and a modified proviral DNA of endogenous viruses present in radiation- or chemically-induced lymphomas, and that an interaction or synergism of MCF and ecotropic viruses is required for MCF virus to exert lymphomagenic activity.

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The mechanism whereby bile acids promote colon tumor development was studied. Bile acids increase intestinal ornithine decarboxylase (ODC), an effect that is suppressed by indomethacin, an inhibitor of prostaglandin (PG) synthesis. Male Sprague-Dawley rats were pretreated with 0.002% indomethacin solution in drinking water for 3 days, then given a single intrarectal instillation of 20mg of deoxycholate and/or 1mg of PGE₂. Four hours later, the rats were killed, and the ODC activity was measured in the mucosa of the distal large bowel. ODC was significantly lower in rats given indomethacin plus deoxycholate than in those given deoxycholate alone, but it was significantly higher in rats treated with indomethacin and PGE₂ plus deoxycholate. Without deoxycholate, indomethacin plus PGE₂ did not elevate ODC compared with indomethacin alone or no treatment. Indomethacin reduced the colonic mucosal PG level. Thus, PGE₂ mediates the deoxycholate-induced colonic mucosal ODC activity, and overcomes the inhibition of this enzyme activity by indomethacin. It is concluded that the anti-promoting effect of indomethacin in colon carcinogenesis, previously demonstrated, may result from the indomethacin inhibition of PG synthesis.

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The human monocytic leukemia cell line, THP-1, is induced to differentiate into more functionally mature monocyte (macrophage)-like cells by incubation with retinoic acid at concentrations of 10nM or higher. There is no apparent morphological change accompanying this functional maturation. These induced cells show increases in nitroblue tetrazolium reduction, immunoerythrophagocytosis, hexose monophosphate shunt activity, and 5'-nucleotidase and NAD⁺-glycohydrolase activities. Prostaglandin E₂, dibutyryl cyclic adenosine 3':5'-monophosphate, or T-lymphocyte-derived differentiation-inducing activity, all inactive or less active alone, increase the extent of differentiation of THP-1 in combination with 10nM retinoic acid. THP-1 is also induced to differentiate by 0.1nM or higher concentrations of cholera toxin. Furthermore, 24, 24-difluoro-1α, 25-dihydroxyvitamin D₃ induces less differentiation of THP-1 compared to retinoic acid. Dimethyl sulfoxide and 12-O-tetradecanoylphorbol-13-acetate show no induction of functional differentiation. THP-1 thus joins the list of leukemic myelomonocytic cell lines (e.g., the promyelocytic HL-60 and the monoblast-like U-937) that are blocked at a relatively late stage of maturation and which differentiate in response to retinoic acid.

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Inoculation of BMA1 cells into BALB/c nude mice formed tumors (BMA1 tumor) that were transplantable into ddY mice, and induced marked granulopoiesis in vivo. Histological study revealed that the tumor was a fibrosarcoma, some parts of which were calcified, and consisted of hemopoietic foci surrounded by adipose tissue. This tumor was regarded as producing CSF in vivo as well as in vitro, since CSF activity was detected in sera of the tumor-bearing mice and tumor extract. Granulopoiesis and splenomegaly developed, associated with an increase of stem cells in the spleen. The number of CFUc and CFUs in the spleen increased to about 91 times and 21 times those of control mice, respectively, whereas the number of stem cells in the tibia did not change significantly. The number of peripheral leukocytes increased to 15 times that of normal mice and amounted to 78% of matured granulocytes. After tumor resection these hematological changes were reversed. The findings suggest that the granulopoiesis in BMA1 tumor-bearing mice may be induced by CSF produced by BMA1 tumor and that the spleen may be a direct target organ of the excessive amount of CSF.

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Chromosomes were successfully studied in 8 of 9 human neuroblastomas (NBs) xenografted in nude mice. Structural abnormalities in the short arm of chromosome #1 were found in 6 of the 8 tumors; these included nonreciprocal translocations and simple deletions. The breakpoints were distributed between 1p11 and 1p34, and all of them had lost the terminal portion of 1p (1pter→1p34). Double minutes (DMs) and homogeneously staining regions (HSRs) were observed in 7 tumors; 6 had either DMs or HSRs, and one had some cells with DMs and other cells with HSR. Only one tumor had neither DMs, nor HSRs. Our study revealed that structural abnormalities in the NB xenografts were essentially the same as those in the NB cell lines and fresh tumors reported previously, and that DMs and HSRs were seen in most NB xenografts as frequently as in NB cell lines.

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Two continuously growing in vito cell lines, PB-1049 and LN-1049, were established from a patient with Ph¹-positive chronic myelogenous leukemia in extramedullary blastic crisis. PB-1049 was established from Epstein-Barr virus (EBV)-infected peripheral lymphocytes of the patient and had a 46, XY, t(9;22) karyotype. This cell line was shown to be tumorigenic in nude mice, and cultured cells recovered from the tumor nodule showed a 46, XY, 6p+, t(9;22) stem line karyotype. LN-1049 was derived from a lymph node culture of the same patient, and was found to be non-tumorigenic in nude mice and karyotypically normal. Immunological examinations on EBV-induced antigens, surface and cytoplasmic immunoglobulin, and monoclonal antibodies, and some enzymatic and electron microscopic studies revealed that both cell lines had attained differentiation into late-B cell stage.

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Selenium concentrations in whole blood of Japanese and American women with and without breast cancer and benign fibrocystic breast disease were determined. The observed blood Se levels of healthy Japanese women (0.286±0.021μg/ml) were similar to previously reported values for healthy Japanese adults. The Japanese patients with benign breast disease and with breast cancer exhibited blood selenium concentrations of 0.200±0.045 and 0.195±0.057μg/ml, respectively. The mean blood Se concentration of Japanese breast cancer patients with recurrence was 0.188±0.061μg/ml. The mean blood Se concentrations of healthy American women from San Diego, Calif., were 0.191±0.023μg/ml; of women with benign fibrocystic disease, 0.142±0.010μg/ml; and of breast cancer patients, 0.167±0.032μg/ml. The higher blood Se concentrations of Japanese healthy subjects as compared to healthy Americans can be attributed to differences in the dietary Se intakes; low blood Se concentration may be indicative of increased breast cancer risk.

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A unique cytotoxic substance (CTS-51), which is rather specific to malignant cell culturesin vitro, was found to be produced together with human immune interferon and interleukin-2 in huffy coat cultures stimulated with staphylococcal enterotoxin B. CTS-51, which is stable at 100° and has a molecular weight of 8, 000-10, 000, was found to have biological properties distinct from those of known cytotoxic cytokines. CTS-51 was able to kill four human malignant cell lines at concentrations that did not affect normal or non-malignant cell lines. The mode of CTS-51 action was found to be cytotoxic rather than cytostatic. Several kinetic studies showed that the cell killing activity seemed to be dose-dependent, and the appearance of the activity required about 24hr. The minimum effective exposure time of the target cells to CTS-51 also seemed to be dose-dependent.

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Seven hybridoma clones producing monoclonal antibodies (HBJ8, HBJ27, HBJ67, HBJ71, HBJ98, HBJ104 and HBJ127) were selected from hybridomas prepared by fusion between P3×Ag8.653 mouse myeloma cells and spleen cells of a BALB/c mouse immunized with T24 human urinary bladder cancer cells, and the binding specificity of, and the molecular characters of the antigens defined by, these monoclonal antibodies were examined. The cell-surface antigens detected with these monoclonal antibodies from T24 bladder cancer cells were as follows: 1) HBJ27-defined gp85 antigen common in all human cells or tissues tested, 2) HBJ98- or HBJ127-defined gp125 antigen distributed in all epithelial and non-epithelial human tumor cell lines tested and in basal layers of the skin or esophagus, proximal tubules of the kidney, and crypts of the gastric and intestinal mucosa, 3) HBJ8-defined gp(40/90) and HBJ67-defined gp83 antigens distributed in a characteristic portion of epithelial tumor cell lines, 4) HBJ71-defined antigen of protein nature and HBJ104-defined antigen of unknown character, both being detected from immunizing T24 cells and a few epithelial tumor cell lines. All these monoclonal antibodies could bind with certain portions of fresh bladder cancer tissues from patients.

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In order to investigate the role of T cells in the production of tumor necrosis factor (TNF), a reconstitution experiment was performed with nude mice (Balb/c, nu/nu). The results obtained were as follows: 1) The cytotoxic activity of tumor necrosis serum (TNS) from Balb/c, nu/nu mice treated with Propionibacterium acnes-LPS was 1/22 of that from Balb/c, nu/+ mice. 2) TNF activity increased 14 times in reconstituted nude mice as compared to Balb/c, nu/nu mice. 3) The production of the cytotoxic activity per cell was investigated using T cell and macrophage fractions separated from the spleens of both Balb/c, nu/nu and Balb/c, nu/+ mice treated with P. acnes as a priming agent. Elicitation with LPS was done in vitro. Release of cytotoxic activity into the culture medium was observed in the macrophage fraction, but not in the T cell fraction. However, no significant species difference was found. 4) With P. acnes treatment, the population of macrophages in the spleens from Balb/c, nu/+ mice increased 25.5 times, whereas that from Balb/c, nu/nu mice only increased 6.8 times. The above results suggest that the mechanism of the incremental effect of T cells on TNF production was due to the promotion of macrophage proliferation during the priming period after injection of P. acnes.

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An immunogenetic evaluation of the antitumor activity of the immunopotentiator Nocardia rubra cell-wall skeleton (N. rubra-CWS) has been performed using non-virus-producing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV) in B10 congenic and recombinant mice. Live tumor cells mixed with either N. rubra-CWS or a placebo control were inoculated intradermally into the right flank of syngeneic mice. With N. rubra-CWS, the development and growth of B10 mouse tumors, S1018(B10) and B1OSA2F, in B10(H-2^b) mice were completely inhibited in all test mice. B10.A(5R) mouse tumor, S322(5R), was completely suppressed in 23 of 25 B10.A(5R)(H-2ⁱ⁵) mice, and B10.BR mouse tumor, S623(BR), was completely suppressed in 7 of 14 B10.BR(H-2^k) mice. However, B10.D2 mouse tumor, S908(D2), in B10.D2(H-2^d) mice and B10.A mouse tumors, S826(BA) and B10ABr1F, in B10.A(H-2^a) mice were not suppressed at all by N. rubra-CWS. N. rubra-CWS showed a remarkable antitumor effect in B10 mice, not only in mixed inoculation with the tumor cells but also with intratumoral administration. These effects were not seen in B10.A mice. Peritoneal exudate cells (PEC) from B10 and B10.A strains previously treated intraperitoneally with N. rubra-CWS and/or MMC-treated tumor cells showed cytolytic and cytostatic activities on tumor cells derived from both strains. These results did not reflect the strain difference seen in the in vivo studies. Mitogenic activity of N. rubra-CWS on spleen cells, however, was significantly different among B10 congenic mice; N. rubra-CWS induced greater blastogenesis of spleen cells from B10 mice than of those from B10.A mice, corresponding to the in vivo results showing strain difference. These results suggest that the host's immunogenetic background may play an important role in cancer therapy with immunopotentiators.

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The effects of adriamycin (AM) on the generation of cell-mediated cytotoxicity in primary stimulation cultures of human peripheral blood mononuclear cells (PBM) with B-lymphoblastoid cell line Raji were assessed. When AM was added to the culture on day 1 for 24hr at a concentration of 10^-8M, the cell-mediated cytotoxic response was significantly augmented as compared to that in untreated culture. The augmented cytotoxic response was significantly depressed when unfractionated cells harvested from untreated culture were added to the effector cells from AM-treated culture. The response was slightly more reduced by addition of cells from untreated culture after treatment with OKT₃. However, the addition of cells from untreated culture after removal of adherent cells resulted rather in an increase of the cytotoxic response. When adherent cell fraction was removed from the effector cells, the cytotoxic response in nonadherent fraction from AM-treated culture was similar to that observed in unfractionated cells from the same culture. On the other hand, nonadherent fraction from untreated culture gave an increased cytotoxic response, which was of almost the same magnitude as that of cells from the augmented, AM-treated culture. These results suggested that the augmentation of cytotoxic response observed in AM-treated culture might be due to inhibition by AM of the generation of suppressive activity in the adherent cell population during PBM-Raji cell culture.

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A phase II study of cis-diamminedichloroplatinum (CDDP, 80mg/m², every 3 weeks) was performed in patients with non-small cell lung cancer (NSCLC). The overall response rate to CDDP was 14% (6/42). In patients without prior chemotherapy, the response rate was 20% (2/10), and in patients with prior chemotherapy, the response rate was 13% (4/31). The major side effect was gastrointestinal toxicity. It was concluded that CDDP at a dose of 80mg/m² every 3 weeks is effective against NSCLC.

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