A highly atypical intraepithelial proliferation, which was interpreted as carcinoma in situ, developed in the urinary bladder of 2 beagle dogs after repeated intravesical instillations of N-methyl-N'-nitrosourea followed by oral administration of N-butyl-N-(4-hydroxybutyl)nitrosamine. The latent period was 37 weeks. The technique may provide a useful model to study the natural history of bladder cancer, particularly carcinoma in situ.

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To investigate the alterations of phosphoseryl/phosphothreonyl-protein phosphatases in neoplastic tissues, the cytosols of rat liver and AH-13, a strain of rat ascites hepatoma, were chromatographed on DEAE-cellulose and the fractions obtained were assayed for protein phosphatase with glycogen synthase D and phosphorylase aas phosphoprotein substrates. While the glycogen synthase phosphatase and phosphorylase phosphatase activities of liver cytosol were largely due to phosphatases IA and II, respectively, as previously reported, these phosphatases were absent or present in only small amounts in AH-13 cytosol, whose glycogen synthase phosphatase and phosphorylase phosphatase activities were due almost wholly to a novel protein phosphatase that appeared to be absent in liver. This phosphatase, termed phosphatase H, was purified further by aminohexyl-Sepharose-4B and Sephadex G-200 chromatography without altering its glycogen synthase D/phosphorylase a activity ratio. Purified phosphatase H required Mg²⁺ or Mn²⁺ for activity and had a molecular weight of about 330, 000. It displayed a substrate specificity broader than that of either phosphatase IA or II.

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^99mTc-labeled L-cysteine, DL-homocysteine, and S-carbamyl-L-cysteine were prepared and tested for effectiveness in gamma camera imaging and for bioradiodistribution in mice bearing Ehrlich solid tumors. Among these labeled compounds, ^99mTc-DL-homocysteine seems most promising for the nuclear medical imaging of tumors.

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Soft agar colony-forming activity of transforming growth factors (TGFs) was examined by a simplified method of soft agar micro-assay using nontransformed mouse BALB/3T3 and normal rat kidney (NRK) cells. Bio-Gel P-60 chromatography of acid-ethanol extracts from a human placenta, a human tumor, and hamster embryos gave similar patterns of colony-forming activity towards BALB/3T3 cells and epidermal growth factor (EGF)-potentiated NRK cells, although the molecular weight ranges of the active fractions were different among the three sources. In the soft agar assay using BALB/3T3 cells, colony-forming activity of a TGF preparation from human placenta was markedly enhanced by insulin, but not by EGF. In contrast, the activity was enhanced by EGF, but not by insulin, when NRK cells were used. Fibroblast growth factor and platelet-derived growth factor induced colonies of both BALB/3T3 and NRK cells, and the induction was suppressed by the TGF preparation. TGF preparations could not induce soft agar colonies of C3H/10T1/2 cells or human foreskin diploid fibroblasts, although they increased the saturation densities of these cell types in monolayer cultures.

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Chicken helper factor (chf)-negative chick embryo cells (CEC) were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cultured at 41° in a CO₂-incubator and are currently in the 400th passage (over 4 years). They were designated as CHCC-OU1. They pile up, and form colonies in soft agar, but do not produce tumors either in the syngeneic chicken or in nude mice. Chf-negative CEC without MNNG treatment were maintained in the same way and are currently in the 350th passage (over 3.5 years). They were designated as SPCC-OU1. They appear normal and form no colony in soft agar. Both cell lines produce avian endogenous leukosis virus of subgroup E.

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A spontaneous tumor was found in the cardiac region of the stomach of an aged orang-outang (Pongo pygmeans) in the Ueno Zoological Garden. This tumor, which was 10cm in diameter and had invaded the serosa, was diagnosed as a squamous cell carcinoma. Metastases were found in the lymph nodes at various sites and in the liver, spleen and bronchus.

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The interspecies cross-reactive melanoma antigen recognized by a syngeneic monoclonal anti-B16 melanoma antibody (M2590) was characterized. The melanoma antigen with cross-species determinants was detected in culture supernates or NP-40 extracts of all melanoma cell lines of mouse, hamster and human origins tested, but not in those of other tumors or normal tissues. Physicochemical analyses using gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the melanoma antigen was a macromolecular complex composed of acidic sialoglycoproteins with a molecular weight of 31, 000 daltons. Moreover, the antigens from mouse and human melanoma were shown to have the same molecular weights. The carbohydrate nature of the cross-species determinant was also investigated by treatment with exoglycosidases or pronase; the results suggested that the epitope is composed of carbohydrate side chains with terminal sialic acid.

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A sensitive sandwich radioimmunoassay for the detection of minute amounts of melanoma antigens from various mammalian melanomas was established by using a syngeneic mouse monoclonal anti-melanoma antibody (M2590). The assay system detected the soluble mouse and human melanoma antigens equally at a concentration of 10³-10⁴cells/ml because the M2590 antibody was found to react specifically with cross-species melanoma antigenic determinants composed of the carbohydrate side chains of the 31, 000-dalton glycoprotein uniquely expressed on various mammalian melanoma cells. Furthermore, human melanomas of various types, such as melanotic, amelanotic, primary, metastatic etc., were successfully detected in an antigen-specific manner. Therefore, the present sandwich assay system using a single monoclonal antibody should be quite useful in clinical applications, especially in the diagnosis of human melanomas.

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The heterogeneity of a tumor antigen TA-4 of squamous cell carcinoma was studied by using isoelectric focusing (IEF). IEF of squamous cell carcinoma tissue of the uterine cervix showed 2 major peaks of TA-4 with pI values of 5.9 to 6.2 (acidic TA-4) and 6.3 to 6.6 (neutral TA-4). The non-malignant squamous epithelium of the uterine cervix showed a major TA-4 peak at pI 6.3 to 6.6 (neutral TA-4), while TA-4 in the blood of patients with squamous cell carcinoma was mainly eluted at pI 5.9 to 6.2 (acidic TA-4). These results indicated that acidic TA-4 is implicated in the appearance of this antigen in the circulation of patients with squamous cell carcinoma.

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Autologous mixed lymphocyte tumor cell culture (MLTC) induced cytotoxic lymphocytes against autologous tumor cells in 4 out of 14 donors (29%). Further cultivation of MLTC-activated lymphocytes with T cell growth factor (TCGF) resulted not only in the propagation of cytotoxic T lymphocytes (CTL) and an increase of cytotoxicity against autologous tumors, but also in the induction of killer cells against allogeneic tumor cells. Briefly, the results of crisscross tests using fresh tumor cells from 17 donors and lymphocytes from 10 donors indicate that cultivation of MLTC-activated lymphocytes with TCGF generated killer cells against allogeneic tumor cells in most cases, but not against autologous or allogeneic phytohemagglutinin-induced lymphoblasts. These effector cells were mainly OKT3⁺, OKT8⁺, OKM1^- and Leu7^-. Further, the results of cold target inhibition tests undertaken in an autologous tumor killing system suggest that at least 2 different subsets, specifically cytotoxic for autologous tumors and cytotoxic for both autologous and allogeneic tumors, were developed in the CTL induced by autologous MLTC followed by cultivation with TCGF.

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Cell assembly at sites eliciting the delayed hypersensitivity reaction (DHR) to tumor antigens was studied using ⁵¹Cr-labeled cells. C3H/He mice were strongly immunized with syngeneic MM46 tumors. The MM46 antigen fraction elicited tumor-specific DHR when injected into the footpad of MM46-immunized mice. The test mice were then injected iv with ⁵¹Cr-labeled spleen cells from normal mice, their feet were cut off, and radioactivity in the feet was counted in a γ-scintillation counter as a measure of the assembly of ⁵¹Cr-spleen cells at the site of the antitumor DHR. Assembly of spleen cells persisted for 48hr after injection of the antigen fraction. Local passive transfer of the DHR also induced assembly of spleen cells, indicating that it is a cell-mediated immune reaction. On the basis of these results, the role of cell movement in host defense systems against tumors is discussed.

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A spontaneous fibrosarcoma (SP/T-1) which arose in a syngeneic Balb/c mouse failed to show clear evidence of immunogenicity when examined by the in vivo immunization challenge technique. However, when the lymph node cells (LNC) of tumor-‘immunized’ donors were cultured in vitro for approximately 2 days in the absence of tumor cells, they were found to be markedly inhibitory to the tumor in cell transfer assays. A similar effect was also found in the LNC of tumor-bearers but this was less marked. The antitumor activity appeared to be mediated largely by the T cells, since the depletion of Thy 1 positive cells abolished or markedly decreased the LNC inhibitory activity towards the tumor cells. The activated LNC were found to be specifically cytotoxic to the SP/T-1 cells since they did not destroy cells of two other syngeneic cell types tested - a methylcholanthrene-induced fibrosarcoma, MC677 and a neonatal heart-derived normal cell line NEO/H. Neither of these cell types showed virus particles by electron microscopy and since C type virus-like particles were detected within the SP/T-1 cells by electron microscopy the possibility exists that viral antigenic determinants expressed on the tumor cell surface acted in tumor cell recognition and destruction in this tumor system. However, the possibility cannot be excluded that in vitro-activated natural killer cells also participated in tumor cell killing. These observations clearly indicate the existence of tumor-associated transplantation antigens in certain ‘non-immunogenic’ tumors as well as antitumor effector mechanisms which remain totally suppressed in vivo.

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The local antitumor activities and inflammation-inducing activities of various antitumor polysaccharides were examined and the relation between the two types of activity was studied. The tested antitumor polysaccharides included MG (a mannoglucan prepared from the culture fluid of Microellobosporia grisea), lentinan, bacterial lipopolysaccharide, TAK (a glucan from Alcaligenes faecalis) and their derivatives. Local antitumor activity was tested by intratumoral administration of the polysaccharides 4, 7 and 10 days after inoculation of MH134 hepatoma intradermally (id) into the abdomen of C3H/He mice. MG and its derivatives showed strong local antitumor activity. Inflammation-inducing activity was assayed by measuring footpad swelling and accumulation of iv-injected ⁵¹Cr-labeled spleen cells after injection of the test materials into the footpads of C3H/He mice. TAK had the strongest inflammation-inducing activity among the polysaccharides tested. No close correlation was found between the local antitumor activity and the inflammation-inducing activity.

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