Induction of tumouricidal macrophages by MTP-PE: its enhancement by a factor produced spontaneously by tumour cells

Tumouricidal activity of rat alveolar macrophages is induced by MTP-PE in vitro. This tumouricidal activity is enhanced by a factor (tumour cell derived immunostimulating factor, TCIF) contained in tumour cell culture supernatants. TCIF is not species specific, since culture supernatants of rat MADB-200 as well as mouse B16 or Meth A tumour cells showed similar effects on rat alveolar macrophages. TCIF is not produced in cultures of normal cells, e.g. rat embryo cells. TCIF produced by MADB-200 tumour cells is relatively heat-stable and dialyzable. It is destroyed by treatment at pH 2 for 24 hrs. These results suggest that TCIF can participate in macrophage activation and could be of potential therapeutic value.     

Abnormal deposition of basement membrane and connective tissue components in dimethylbenzanthracene-induced rat mammary tumours: an immunocytochemical and ultrastructural study

Summary Dimethylbenzanthracene-induced rat mammary tumours consist of lobules of tumours cells surrounded by connective tissue. The interstitial connective tissue proteins, collagen types I, III and V, fibronectin and elastin are largely restricted to the interlobular connective tissue. The tumour lobules are surrounded by a basement membrane that stains with antiserum to laminin. Electron microscopy reveals a greatly thickened basement membrane to which striated interstitial collagen fibres are closely juxtaposed. The lumina within the tumour lobules are of two types. In the first type, the luminal surface is characterized by the presence of microvilli and tight junctions are reacts with antiserum to rat milk fat globule membrane. In the second type, the luminal surface is flattened and lined by a thickened basement membrane that stains with antiserum to laminin and type IV collagen. These abnormal patterns of growth and differentiation may be partly a consequence of the disorganization of extracellular matrix components at the interface between the tumour epithelial cells and the surrounding stroma.     

Characterization of Catecholamine-Storage Organelles in Transplantable Phaeochromocytoma and Adrenal Glands of Rats

Abstract: The properties of the catecholamine-storing organelles from transplantable rat phaeochromocytoma and rat adrenal glands were compared by density gradient centrifugation. It was shown that tumour granules are more heterogeneous and less dense than adrenal granules. Both granule preparations can take up catecholamines and nucleotides by a process driven by an electrochemical proton gradient. Dopamine β-hydroxylase and glycoprotein III were analysed by immunological techniques. Glycoprotein III was shown to be a specific component of chromaffin granules. Tumour tissue (average weight 700 mg) contains amounts of these antigens comparable to those in 210 adrenals. The biosynthesis of granules in the tumour apparently occurs at a low rate, making turnover studies difficult. The transplantable rat phaeochromocytoma is very useful for studies on the uptake properties and the immunological characteristics of rat catecholamine storage granules because one tumour provides an amount of material that could otherwise be obtained only from a large number of adrenal glands.     

Hetero-organic antigens of renal nature in the composition of the nuclear nonhistone proteins in the regenerating rat liver

In the nuclear non-histone proteins of the rat liver, on the 4th day after a partial hepatoectomy or hepatocarcinogen injection as well as in the hepatocellular tumour cells, some heteroorganic antigens of kidney nature (HAkid) are found and characterized immunochemically. These HAkid can be eluted at 0.4-0.5 M NaCl during gradient chromatography on phosphocellulose. They possess some proper phosphoproteinkinase activity. The appearance of HAkid in rat liver may be considered, on the one hand, as a manifestation of some malignant factor (carcinogen action, tumour cells) and, on the other hand, they are obviously connected with proliferative activity of the regenerative rat liver cells (hepatectomy effect). It is very likely that both the sides of this phenomenological effect of HAkid in rat liver are the consequence of a specific expression along the cell oncogens.     

Intermediate cell tumour of the pancreas in rat

A malignant tumour of the rat pancreas with features of both acinar and endocrine cells is presented. This consisted of a continuous cytoplasmic mass with numerous dispersed nuclei and branches protruding from its borders invading the surrounding exocrine tissue. The most prominent characteristic of the tumour was the co-existence of zymogen and endocrine secretory granules and cytoplasmic organelles typical of both acinar and islet cells. Some hypotheses are put forward concerning the origin of the tumour and its vasculature.     

Effects of polyamines on DNA synthesis using various subcellular DNA polymerases extracted from normal rat liver, tumour-bearing rat liver, and tumour cells

The effects of polyamines on DNA synthesis in vitro using various subcellular DNA polymerase fractions from normal and tumour-bearing rat livers, and tumour cells were investigated. When nuclear and mitochondrial DNA polymerase fractions were used, DNA synthesis on activated DNA was increased 3.5-8-fold by the addition of 20 mM putrescine or cadaverine. However, DNA synthesis was not stimulated by the addition of spermidine or spermine at any concentration tested. In contrast, DNA synthesis using the cytoplasmic DNA polymerase fraction was not stimulated at various concentrations of any of the four polyamines tested. The stimulatory effects of putrescine and cadaverine were absent when nuclear fractions from tumour-bearing rat liver or from tumour cells were used. In addition, in vitro DNA synthesis was not stimulated by 20 mM putrescine or cadaverine when nuclear extracts from the livers of rats administered putrescine subcutaneously were used. The specific activities of DNA polymerases extracted from tumour cells and tumour-bearing rat liver were already fully stimulated. These results suggest that DNA polymerases in tumour cells and tumour-bearing liver cells are stimulated by trapped putrescine produced in tumour cells and are thus no longer activated by exogenous putrescine.     

Differential phosphodiesterase expression and cytosolic Ca2+ in human CNS tumour cells and in non-malignant and malignant cells of rat origin

A promising attempt in the field of tumour therapy is the modulation of intracellular, proliferation-associated signalling pathways. The role of cyclic nucleotide phosphodiesterases (PDEs), key enzymes in cAMP/cGMP signal transduction, was investigated in two human CNS tumour cell lines as well as in the rat glioblastoma cell line C6 in comparison with rat cerebellar astrocytes with the emphasis on target evaluation. We found differential PDE expression patterns in human CNS tumour cell lines as well as in CNS cells of rat origin. In human glioblastoma cells, intracellular cAMP and Ca(2+) levels correlated well with the PDE expression pattern. There were, however, marked differences in PDE expression and Ca(2+) kinetics between the human glioblastoma cell lines. In contrast to human epithelial tumour cells, shown earlier by us to express significantly enhanced cAMP-specific PDE activity, this was not the case in rat glioblastoma cells compared with non-malignant rat astrocytes. Despite different levels of PDE1 and PDE4 expression and activity, cyclic nucleotide and Ca(2+) levels in non-malignant and malignant rat CNS cells were similar. These in vitro data do not support the concept of PDE1C representing a target exploitable for drug treatment of malignant CNS tumours.     

Inhibition of rat yolk sac tumour growth in vivo by a monoclonal antibody to the retroviral molecule P15E

Summary A monoclonal mouse IgG2b antibody 19F8, directed towards a determinant on the retroviral transmembranous molecule p15E, binds selectively to certain rat tumours, including all tested yolk sac tumours, one rat colon carcinoma, one spontaneous kidney carcinoma and an adenovirus-type-9-induced rat breast tumour, as tested by antibody-dependent cellular cytotoxicity (ADCC) and immunohistochemistry. Groups of rats receiving yolk sac tumour F56 isografts intraperitoneally (i.p.) or subperitoneally (s.p.) were treated with this monoclonal antibody (mAb), 19F8, inoculated twice a week in doses of 100 µg. Parallel control groups received analogous inoculations of an isotype-matched monoclonal antibody. A significant growth inhibitory effect was observed with 19F8. In 5/10 rats isografted i.p., tumour outgrowth was completely inhibited and in the other 5 rats the outgrowth was delayed compared to the 10 rats in the control group, which all developed tumours. All rats of the control group developed large volumes of ascites, whereas the 5 rats in the therapy group with eventual tumour outgrowth had little or no ascites. In two experiments with rats carrying subperitoneal isografts and treated with the 19F8 mAb, tumour grew out in 4/5 and 5/10 rats, though growth was delayed compared to the control groups, in which 5/5 and 9/9 rats developed tumours. The tumours grew significantly more slowly in the therapy groups compared to the controls. All rats that developed tumours in the therapy groups showed an anti-idiotypic response against mAb 19F8. The single tumour-free rat in the first experiment and 1/5 tumour-free rats in the other showed no such response. The draining lymph node cells from the tumour-free animals showed a specific proliferative response to yolk sac tumour F56 cells in a mixed lymphocyte tumour cell culture (MLTC), and the MLTC-induced cells were cytotoxic to F56 but not to the natural-killer-sensitive rat T cell lymphoma G1—Tc1. The cytotoxic cell population was more than 90% CD4⁺. It is concluded that the two test systems for identification of the epitope of p15E detected by mAb 19F8 correlated well in detection of the epitope in the cells (immunohistochemistry) and at the cell surface (ADCC). It is also concluded that mAb 19F8 has a growth-inhibitory effect on yolk sac tumour F56 and that, as a result of the treatment, T cells with specificity for F56 are appearing in draining lymph nodes of tumour-free animals.     

Comparison of rat liver and Walker 256 carcinosarcoma tRNAs.

The complete nucleotide sequences of both rat liver and Walker 256 mammary carcinosarcoma tRNAAsn reveal that they are identical except for the nucleotide present in the wobble position of the anticodon loop. The rat liver tRNAAsn contains the Q nucleoside, whereas the tumour tRNAAsn contains an unmodified guanosine. The tRNAs from both tissues also show significant quantitative differences in the chromatographic mobilities for isoaccepting species of tRNAAsp, tRNAAsn, tRNAHis and tRNATyr. In addition, chromatographic shifts upon cyanogen bromide treatment and analyses of the alkaline hydrolysates of these tRNAs demonstrate that those of tumour origin contain significantly less Q and Q nucleoside than their normal rat liver counterparts.     

Bone remodelling and tumour grade modifications induced by interactions between bone and swarm rat chondrosarcoma

Chondrosarcoma is currently defined as a malignant cartilage tumour arising de novo or within a pre-existing benign cartilage tumour. Chondrosarcoma can be surgically resected, but all grades have significant rates of local recurrence. The purpose of the present study was to develop an animal intraosseous chondrosarcoma model simulating the progression of human chondrosarcoma and elucidating its behaviour and biology. An intraosseous Swarm rat model was designed to assess interactions between bone and chondrosarcoma. A comparison of tumour grading was carried out according to transplantation site. The effects of chondrosarcoma cells (SRC cells) on the mineralisation capacities of osteoblasts and on osteoclast differentiation were studied in relation to modifications observed in vivo at the cellular level. Transplantation of Swarm rat chondrosarcoma within bone marrow or contiguous to induced periosteal lesions led to extensive bone remodelling with trabecular bone rarefaction and periosteal apposition. Transplantation in close contact to bone but without any periosteal lesion had no effect on bone, suggesting that bone healing factors interact with tumour development. With the intramedullary model, the development of tumours of different grade confirms that bone environment is an important factor in malignancy. A decrease of bone nodule formation was noted after cocultures of SRC cells with rat bone marrow, but there was no modification of osteoclast differentiation after cultures of total rabbit bone cells with SRC cells. These data reveal the importance of interactions between bone environment and tumour in inducing bone remodelling and variations in tumour malignancy.     

A Fourier transform infrared microspectroscopic imaging investigation into an animal model exhibiting glioblastoma multiforme

Glioblastoma multiforme (GBM) is a highly malignant human brain tumour for which no cure is available at present. Numerous clinical studies as well as animal experiments are under way with the goal being to understand tumour biology and develop potential therapeutic approaches. C6 cell glioma in the adult rat is a frequently used and well accepted animal model for the malignant human glial tumour. By combining standard analytical methods such as histology and immunohistochemistry with Fourier Transform Infrared (FTIR) microspectroscopic imaging and multivariate statistical approaches, we are developing a novel approach to tumour diagnosis which allows us to obtain information about the structure and composition of tumour tissues that could not be obtained easily with either method alone. We have used a “Stingray” FTIR imaging spectrometer to analyse and compare the compositions of coronal brain tissue sections of a tumour-bearing animal and those from a healthy animal. We have found that the tumour tissue has a characteristic chemical signature, which distinguishes it from tumour-free brain tissue. The physical-chemical differences, determined by image and spectral comparison are consistent with changes in total protein absorbance, phosphodiester absorbance and physical dispersive artefacts. The results indicate that FTIR imaging analysis could become a valuable analytic method in brain tumour research and possibly in the diagnosis of human brain tumours.     

A Fourier transform infrared microspectroscopic imaging investigation into an animal model exhibiting glioblastoma multiforme

Glioblastoma multiforme (GBM) is a highly malignant human brain tumour for which no cure is available at present. Numerous clinical studies as well as animal experiments are under way with the goal being to understand tumour biology and develop potential therapeutic approaches. C6 cell glioma in the adult rat is a frequently used and well accepted animal model for the malignant human glial tumour. By combining standard analytical methods such as histology and immunohistochemistry with Fourier Transform Infrared (FTIR) microspectroscopic imaging and multivariate statistical approaches, we are developing a novel approach to tumour diagnosis which allows us to obtain information about the structure and composition of tumour tissues that could not be obtained easily with either method alone. We have used a "Stingray" FTIR imaging spectrometer to analyse and compare the compositions of coronal brain tissue sections of a tumour-bearing animal and those from a healthy animal. We have found that the tumour tissue has a characteristic chemical signature, which distinguishes it from tumour-free brain tissue. The physical-chemical differences, determined by image and spectral comparison are consistent with changes in total protein absorbance, phosphodiester absorbance and physical dispersive artefacts. The results indicate that FTIR imaging analysis could become a valuable analytic method in brain tumour research and possibly in the diagnosis of human brain tumours.     

Pyruvate kinase isoenzymes in chromatin extracts of Ehrlich ascites tumour, Morris hepatoma 7777 and normal mouse and rat livers

A pyruvate kinase (EC 2.7.1.40) variant inhibited by L-cysteine has been found in Ehrlich ascites tumour and Morris hepatoma 7777, but not in normal mouse and rat livers used for comparison. Chromatin extracts of all materials studied contained three pyruvate kinase isoenzymes (alpha, beta, gamma) which showed the greatest electrophoretic mobility in normal mouse and rat livers. The isoenzyme mobility diminished in both tumour chromatin extracts, and the slow migrating gamma isoenzyme exhibited sensitivity to L-cysteine inhibition. This gamma isoenzyme sensitive to L-cysteine might be considered as a tumour marker. All tumour pyruvate kinase isoenzymes were insensitive to normal signal molecules, i.e., to ATP and fructose 1,6-diphosphate, which regulate liver pyruvate kinase activity. It was, however, noted that the binding of pyruvate kinase isoenzymes to DNA is connected with a diminution in their catalytic activity.     

Rat macrophage activation after treatment with the bleomycin group of antitumour antibiotics in vivo

Summary We have previously reported that bleomycin and its derivative peplomycin enhance the release of cytokines by rat spleen cells during mitogen-stimulated cell culture in vitro, but liblomycin, another derivative of bleomycin, decreases cytokine release to below untreated control levels. Cytokine release correlated well with the inhibition of subcutaneous tumour growth after treatment with equivalent doses of the three analogues. In contrast, ascites tumour growth is completely inhibited by liblomycin and appears to be at least partly macrophage-mediated because the antitumour effect can be significantly inhibited by carageenan. This study shows that bleomycin and its analogues activate rat peritoneal macrophages and increase interleukin-6 release, O₂ ^– production, cell spreading, phagocytosis and random migration of macrophages, but only bleomycin enhances peritoneal macrophage invasion into a monolayer of rat lung endothelial cells in vitro. This study also shows that although liblomycin decreases spleen cell cytokine production and is less effective than bleomycin against subcutaneous tumour, as we have previously reported, the antitumour drug activates peritoneal macrophages and, compared to bleomycin, has a remarkable therapeutic effect on rat ascites tumour.     

Studies on lipid peroxidation in normal and tumour tissues. The Novikoff rat liver tumour.

A study has been made of the factors that contribute to the decreased rates of lipid peroxidation under different pro-oxidant conditions in intact Novikoff tumour cells, and in microsomal suspensions prepared from Novikoff tumour cells, compared with isolated normal rat hepatocytes and microsomal suspensions prepared from normal rat liver. The pro-oxidant conditions were the addition of either NADPH, NADPH + ADP + iron, NADPH + CCl4 or ascorbate+iron to the experimental systems used, or exposure to gamma-radiation. Contributory factors to the lower rates of lipid peroxidation observed include: a significant decrease in the polyunsaturated fatty acid content of Novikoff cells or Novikoff microsomes; the decreases are especially marked for the C20:4 and C22:6 fatty acids; a very marked reduction in NADPH-cytochrome c reductase; and no detectable content of cytochrome P-450. Another, and in our opinion critical, contribution to the diminished rate of lipid peroxidation in the tumour material is the substantial increase in alpha-tocopherol relative both to total lipid and to methylene-interrupted double bonds in fatty acids. Moreover, the alpha-tocopherol is the major contributor to lipid-soluble chain-breaking antioxidant in lipid extracts of normal liver and of Novikoff tumour material.     

A new member of the troponin C superfamily: Comparison of the primary structures of rat oncomodulin and rat parvalbumin

When the amino-acid sequence of the 108-residue, rat tumour calcium-binding protein, oncomodulin, was aligned with that of rat muscle parvalbumin, 55 homologous positions were found, with an additional 33 single base-pair substitutions. This extensive homology, with virtual identity of the calcium-binding domains, signalled oncomodulin to be a member of the troponin C superfamily. The presence of Cys-18 and Phe-66 in oncomodulin, plus its isoelectric point of 3.9, suggest that this tumour protein is a beta-parvalbumin, rather than a muscle alpha-parvalbumin.     

Limited effects of recombinant human and murine interleukin 1 and tumour necrosis factor on production of acute phase proteins by cultured rat hepatocytes

Albumin, fibrinogen, alpha 1-acid glycoprotein and cysteine proteinase inhibitor were determined by electroimmunoassay in the media of primary cultures of rat hepatocytes exposed to dialysed supernatants of rat, mouse and human macrophages or to recombinant human and murine interleukin 1 and tumour necrosis factor. Recombinant cytokines in the range of 1 to 1000 ng/ml caused only reduction of albumin synthesis and slight stimulation of alpha 1 acid glycoprotein production while crude preparations of macrophage cytokines elicited typical acute phase response. The results suggest that interleukin 1 or tumour necrosis factor are not likely the principal mediators responsible for the direct stimulation of normal rat hepatocytes to acute phase protein synthesis.     

Tumour localization of monoclonal antibody against a rat mammary carcinoma and suppression of tumour growth with adriamycin-antibody conjugates

Summary Monoclonal antibody to the rat mammary carcinoma Sp4 isolated from hybridoma supernatants and labelled with ¹²⁵I showed preferential in vivo localization into subcutaneous growths of Sp4 compared with normal tissues and a range of other transplanted tumours. No specific localization was seen with ¹²⁵I-labelled normal rat immunoglobulin. Adriamycin conjugated to monoclonal antibody significantly retarded Sp4 tumour growth at 1/25th of the effective dose of the free drug, and in some cases brought about total tumour regression. Normal rat immunoglobulin-adriamycin conjugates were relatively ineffective. These studies indicate that monoclonal antibody directed against tumour cell surface antigens may be highly effective for tumour targeting of therapeutic agents.     

Rat C-Type Virus induced in Rat Sarcoma Cells by 5-Bromodeoxyuridine

HALOGENATED derivatives of uridine are highly effective inducers of latent C-type RNA viruses^1,2 and have been successfully used to induce viruses identical to, or similar to, the C-type RNA tumour viruses in mouse, rat and human cells^3–6. In previous experiments we used 5-bromodeoxyuridine (BrUdR) for induction of focus-forming virus in non-productive rat cells that have been transformed by mouse sarcoma virus². We describe here the induction of a C-type RNA virus in the cells of the rat tumour cell line XC, which contains the Rous sarcoma virus genome⁷. The induced virus possesses the group specific (gs) antigens of rat C-type viruses but not those of chicken C-type viruses.     

A Leydig cell tumour: a model for the study of lutropin action.

The properties of cells isolated from a Leydig cell tumour have been compared with normal rat testis Leydig cells. These cells were found to be similar in the following respects: 1. Lutropin-stimulated cyclic AMP and testosterone production. 2. Lutropin-activated protein kinase activity followed by phosphorylation of endogenous proteins of mol. wts. 57,000and 14,000. 3. Parallel lutropin dose vs. response curves for phosphorylation of the endogenous proteins and for testosterone production. 4. Two forms of isoenzyme, cyclic AMP dependent protein kinase, present. They differed mainly with respect to the lutropin-stimulated testosterone production, which was much lower in the tumour cells compared with the normal adult testis Leydig cells (4.6 +/- 1.1 and 114 +/- 16 ng testosterone/10(6) cells per 2 h, respectively). However, the lutropin-stimulated steroid production in the tumour cells was quantitatively comparable with the normal rat Leydig cell when the metabolism of pregnenolone in intact cells and mitochondria was inhibited by addition of SU-10603 and/or cyanoketone. It is concluded that the Leydig cell tumour used in this study can be used to investigate certain aspects of lutropin action where large quantities of cells are required.