A transplantable rat liver tumor induced by 4-dimethylaminoazobenzene.

1. A transplantable tumor was established in 1951 from a primary nodule and mesenteric metastases of a DAB-induced liver tumor. After the fourth transplant generation, the tumor grew rapidly as generally non-necrotic masses throughout the peritoneum and mesentery. After 6 months, growth became less widespread, the animals died earlier, and a striking decrease in the number of mucin-producing cells occurred. In another 6 months, the mucinous cells disappeared completely. 2. The tumor disseminates widely by direct invasion and lymphatic spread. 3. Regressions occur rarely, if ever, when the tumor is planted intraperitoneally, except at low doses. However, tumors grown intramuscularly in the hind leg, where they may reach enormous size, regress in about 40 per cent of the cases. Tumors grown subcutaneously regress even more frequently. Animals in which hind leg regression has occurred are totally resistant to tumor growth at any site tested. 4. In preliminary experiments, resistance to growth of transplanted tumor cells has been induced by injection of cell-free microsome fractions. 5. The dimensions, morphology, and chemical and enzymatic composition of cytological structures in the tumor cell are described. This is based on phase-contrast microscopic study of isolated cells in homogenates, light microscopic study of sections stained with cytological and cytochemical procedures, and electron microscopic study of thin sections of osmium-fixed tissue and homogenates. From these studies a diagram of the tumor cell is constructed. 6. The nuclei, large and irregular, contain large nucleoli attached to the nuclear membrane. In electron micrographs, the nuclear envelope consists of two membranes coated with dense granules like those in the cytoplasm. The nucleoli, in parts at least, consist of similar granules arranged linearly on two membranes (nucleolonemata). The membranes of sickle-shaped structures, tentatively considered to be part of or attached to the nucleolonemata, are continuous with the inner nuclear membrane. The chromatin is arranged in clumps near the nuclear membrane and nucleoli and as a thin layer against the inner nuclear membrane. In electron micrographs, it consists of small, dense granules linearly arranged on fine membranes; these are finer than those of the nucleolonemata. The possibility is considered that the granules of the nucleolonemata are composed of ribonucleoprotein, and the smaller chromatin granules of deoxyribonucleoprotein. 7. Electron microscopy reveals that the plasma membrane and the outer nuclear membrane are continuous through parallel double membranes, tentatively labeled “intracellular channels.” Their possible significance in molecular transport is considered. Other cytoplasmic structures are: mitochondria (small, few in number, with typical fine structure, and containing succinoxidase and phospholipide); numerous lipide droplets (containing phospholipide); Golgi apparatus (which shows the same cuplike shape in unfixed cells, in classic Golgi preparations, in sections stained for phospholipide, and in electron micrographs; its fine structure consists of evenly spaced membranes, generally three in number); and small ribonucleoprotein granules (most of which appear free in the cytoplasm and some of which are arranged on the membranes of the “intracellular channels” and outer nuclear membrane). 8. Each tumor cell is exposed in areas to “extracellular channels.” Here the plasma membrane is extended into multiple “microvilli”; these apparently possess ATP-ase activity and contain a mucopolysaccharide. The “extracellular channels” form an anastomosing network which opens to the subendothelial area of the blood vessels. 9. Squashes of cells in metaphase show (Dr. T. C. Hsu) two classes of cells, one with a modal number of 39 and another with 75–78; this includes a long V and a short dicentric chromosome. 10. Chemical analyses of tumor homogenates show a high content of water, RNA, and DNA; and considerable phospholipide and adenylatekinase and inorganic pyrophosphatase activities. No uricase activity and extremely low esterase activities are present. Levels of succinoxidase and alkaline phosphatase activities are low. Dephosphorylation of ATP is stimulated by both calcium and magnesium ions. These properties are compared with those of normal liver and primary tumor homogenates. 11. Chemical and enzymatic properties of subcellular fractions isolated by differential centrifugation are described. Difficulties in interpretation of such data for tumor cells are discussed, and the value of cytochemical stains is stressed. 12. The probable cell of origin of the transplantable tumor is discussed. The histology, histochemical and cytochemical staining reactions, and chemical assays of homogenates and subcellular fractions fail to reveal any definitively hepatic features in the tumor. However, no specific feature is presently known by which to distinguish rapidly growing hepatic tumor from tumors originating in other tissues.