Intraperitoneal transplantation of Yoshida ascites hepatoma cells after fine needle aspiration from tumor bearing rats organs

The authors tested the ability of scattered tumor cells to re-form a tumor in vivo. Disseminated tumor cells are morphologically visible stained with May-Grünwald Giemsa in the lung, liver, kidney, and spleen of Yoshida ascites tumor bearing rats. Free tumor cells can easily be fine needle aspirated from those organs and injected in syngeneic Wistar rats. All the host rats show ascites tumor take after intraperitoneal transplantation of each aspirated sample. This biological model might be useful to study in vivo a wide range of properties of neoplastic and non-neoplastic host cells.     

A Polydeoxythymidylic acid-specific deoxyribonuclease from rat ascites hepatoma cells.

A deoxyribonuclease has been purified 950-fold from rat ascites hepatoma cells and has been separated from another deoxyribonuclease that appears to have DNase III type activity. The enzyme preferentially degrades single stranded poly(dT), requires Mg²⁺ for maximum activity and has a pH optimum at 8.5 in Tris-HCl buffer. Poly(dA), poly(dC), poly(rA), and poly(rU) are not effective substrates. The hydrolysis of poly(dT) is strongly inhibited when poly(dA) or poly(rA) is annealed with poly(dT). Poly(dT) is degraded ultimately into 5′-deoxythymidylic acid via the formation of oligodeoxythymidylate intermediates.     

Reassociation kinetics of DNA from x-irradiated ascites hepatoma cells of the rat.

The kinetics of the reassociation fo DNA from ascites hepatoma cells has been studied. The curve exhibited three zones corresponding to 'fast', 'intermediate' and 'slow' speeds of DNA reassociation. The difference was observed in the DNA reassociation curves of the control and irradiated (1500 rad) cells which was particularly expressed in the 'slow' zone (10(2) less than C0t less than 10(4). The same dose, however, does not qualitatively effect the secondary DNA structure, which was estimated by the method of thermal elution from the hydroxyapatite column.     

Studies on the molecular species of DNA polymerase extracted from rat ascites hepatoma cells.

DNA polymerase [EC 2.7.7.7] activities present in hypotonic extract from rat ascites hepatoma AH130 cells were eluted in three separable peaks on DEAE-cellulose column chromatography. Peak I activity had an alkaline pH optimum, and was relatively resistant to SH-blocking reagents and salt concentration. These properties of DEAE peak I are typical of low molecular weight DNA polymerase. DEAE peak II and peak III activities possessed properties corresponding to high molecular weight (6-8 S) polymerase; they showed maximal activity at neutral pH, and were sensitive to SH-blocking reagents and salt. No low molecular weight polymerase activity was released from DEAE peak II or peak III by salt treatment, though partial conversion from DEAE peak II to peak III was observed on the same treatment.     

Conversion of DNA polymerase extracted from rat ascites hepatoma cells

DNA polymerase extracted fresh from rat ascites hepatoma cells possesses high molecular weight, maximal activity at neutral pH, and high sensitivity to N-ethylmaleimide (NEM). After physical and chemical treatment of the enzyme fraction, the appearance of low molecular weight DNA polymerase was detected by means of Sephadex gel filtration or sucrose density gradient centrifugation. This low molecular weight DNA polymerase possesses alkaline pH optimum, preference of native DNA as template/primer, and relative resistance to NEM.     

Purification and characterization of alkaline phosphatase from cultured rat ascites hepatoma cells.

Alkaline phosphatase of cultured rat ascites hepatoma cells has been purified by butanol extraction, DEAE-cellulose column chromatography, gel filtration through Sephadex G-200, concanavalin A-Sepharose affinity chromatography, and polyacrylamide gel electrophoresis. Affinity chromatography confirmed the glycoprotein nature of alkaline phosphatase from cultured rat ascites hepatoma cells. Electrophoresis on polyacrylamide gels of various concentrations indicated a molecular weight of 290,000. The molecular weight of the subunit was estimated to be 72,000 by SDS-polyacrylamide gel electrophoresis. These findings suggest that alkaline phosphatase of cultured rat ascites hepatoma cells is a tetramer with a subunit molecular weight of 72,000.     

Release of free fatty acids from Ehrlich ascites tumor cells

Ehrlich ascites tumor cells release free fatty acids (FFA) during in vitro incubation in media that contain albumin. The released FFA are derived by lipolysis from endogenous lipid esters. Addition of glucose to the incubation medium greatly decreases the quantity of fatty acid released by the cells. Cyanide, which inhibits endogenous lipid oxidation but not lipolysis, increases the quantity of fatty acid released to media containing albumin and causes free fatty acid to accumulate in the cells in the absence of exogenous albumin. The release of fatty acid, either preformed or derived by lipolysis during prolonged incubations, occurs under conditions of net fatty acid uptake from the incubation medium. Net release of fatty acid from the cell occurs only when fatty acid-extracted albumin is present in the extracellular medium; extrapolation of the data suggests that net release will not occur under physiological conditions. It is postulated that free fatty acid uptake and release are independent processes, the direction of net fatty acid movement being determined by the relationship between cellular free fatty acid concentration (regulating efflux) and the molar ratio of free fatty acid to albumin in the extracellular medium (regulating uptake).     

Internalization of cationized ferritin receptors by rat hepatoma ascites cells.

Cationized ferritin (CF) was used to label the cell surface anionic sites of Chang rat hepatoma ascites cells. If the hepatoma cells were fixed with glutaraldehyde and treated with CF, the label was distributed evenly over the external surface of the plasma membrane. Treatment of unfixed ascites cells with CF yielded clusters of ferritin particles separated by label-free areas of the plasma membrane. Some unfixed ascites cells were treated firstly with CF, then incubated in veronal buffered saline at 37 °C for 10, 20, 30 and 45 min, subsequently fixed in glutaraldehyde and re-exposed to CF. After 10 min of incubation, the label was arranged into large clusters with the remaining areas of the plasma membrane lightly labelled with CF. At 20 min, only clusters of ferritin were present on the plasma membrane; the remaining area of the cell surface was totally free of label. The ability of the plasma membrane to bind additional CF was completely restored after 45 min of incubation. These results suggest that for some period of time after internalization of CF label on cell surface the plasma membrane is devoid of any detectable negative charge.     

Endonucleolytic activities of nuclei from rat ascites hepatoma

By autodigestion (endogenous endonucleolysis) of rat liver (RL) or rat-ascites hepatoma (AH) nuclei, the nucleosomes were released from the RL, but not from the AH, nuclei. In contrast, by micrococcal nuclease digestion (exogenous endonucleolysis), the nucleosomes were released more rapidly from the AH than from the RL nuclei. A 0.6 M NaCl extract of the RL or AH nuclei was filtered through a Sephadex G-100 column. The resulting topoisomerase fraction was subjected to DNA relaxation and catenation assays with pBR322 DNA as a substrate. Consequently, the relaxation activity was almost the same between the RL and AH fractions, whereas the catenation activity was much higher in the AH fraction.     

Glycosaminoglycans and electrokinetic behavior of rat ascites hepatoma cells

Cellular electrophoretic mobility of AH-130, an island-forming strain of rat ascites hepatomas, was reduced by chondroitinase-ABC treatment of cells but not affected by neuraminase. Assay of released sugars demonstrated the presence of chondroitin sulfates at the cell surface of AH-130, indicating that acidic residues of chondroitin sulfates were one of the factors responsible for negative surface charge of these cells and sialic acid was not. Surface-located chondroitin sulfates in AH-130 cells were abundant in chondroitin sulfate A. The mobility of free-cell-type subline cells was also lowered by the chondroitinase as well as by the neuraminidase, indicating the presence of chondroitin sulfates on the cell surface. The mobility of rat erythrocytes, however, was not affected by the chondroitinase.     

Electron microscopic studies on chromatin loop of rat ascites hepatoma cells

Isolated nuclei from rat ascites hepatoma cells were treated with 0.09% detergent Joy and chromatin, protruded from the nucleus, was observed with an electron microscope. It was demonstrated that most, but not all of the protruded chromatin fibers had a loop structure. The protrusion of chromatin from the nucleus was 3 microns in average length. A high magnification view showed that the protruded chromatin consisted mainly of beaded nucleosomal fiber. Therefore, the chromatin loop size at the level of nucleosomal fiber was estimated to be at least 6 microns in length.     

Sialyl transferase activities of rat ascites hepatoma cells and rat liver

Sialyl transferase activities of the homogenate of rat ascites hepatome cells were compared with normal rat liver homogenate. The former had only 20% of the activity of the latter when an exogenous acceptor was added in the reaction mixture.Toward endogenous receptors, the activity of the hepatoma cell homogenate was 50% of that of the normal cell homogenate. A stimulation of the activity toward endogenous acceptors was observed when the homogenate of rat ascites hepatoma cells and that of rat liver were mixed. This stimulatory effect seems to be the consequence of utilization of acceptors from ascites hepatoma cells by the sialyl transferases of the rat liver.     

Morphological and functional heterogeneity of Zajdela rat ascites hepatoma cells

This work studies the mechanisms of dysdifferentiation at cell neoplastic transformation based on the example of heterogeneity of the cell populations that form malignant tumors. Two natural fractions of Zajdela rat hepatoma cells are revealed that differ in the type of growth in the primary culture. Cells of one fraction are attached to substrate and are growing in monolayer (S-fraction), whereas cells of the other fraction are floating in the culture medium (F-fraction). Using the method of supravital observation of the primary culture cells (of 1–2 passages) at the limit of resolution of DIC microscopy, it has been established that both fractions contain cells of several types. Some of these cells are specific to one of the fractions and others are present in both fractions, but with different frequencies. Using the same method, it has been shown that, at the long-term separate cultivation of the fractions in vitro (more than 50 passages), both the cell composition and the initial ratio of cells of different types are changed in both of them. According to the data of flow DNA cytometry, cells of both fractions are hypotetraploid and have insignificant differences in the amount of DNA. After adaptation to conditions of cultivation in vitro, S-fraction cells have been found to have elevated proliferative activity compared to the cells of F-fractions; after long cultivation, the fractions already differ significantly (2.3 times) by this criterion. The content of the cell surface laminin, a marker of hepatocellular carcinomas, is higher on cells of the F-fraction than on those of the S-fraction. The interfraction differences are confirmed by immunologic estimations of the resistance of hepatoma cells to lyses of natural killer cells; cells of the S-fraction of the primary culture are 2.4 times more sensitive than cells of the F-fraction, while, after long-term cultivation, cells of the F-fraction become almost resistant to the cytotoxic action of natural killer cells. Based on the obtained data, the most probable pathways of the dysdifferentiation of rat hepatocytes upon the establishment of Zajdela hepatoma and at the long-term cultivation of cells of this tumor in vitro are discussed.