Hepatoma AH109A Research Articles

Studies on responsiveness of hepatoma cells to catecholamines. II. Comparison of beta-adrenergic responsiveness of rat ascites hepatoma cells with cultured normal rat liver cells.

The pharmacological properties of beta-adrenoceptors in rat ascites hepatoma cells were compared with those in normal rat liver cells which were cultured for 24 hr after collagenase digestion. Adenylate cyclases in the homogenates of cultured normal rat liver cells and rat ascites hepatoma cells, AH44, AH66, AH109A, AH130 and AH7974, were all activated by isoproterenol or NaF to different degrees. The enzyme in rat liver cells was activated by several beta 2-agonists but those in all hepatoma cells hardly responded. Furthermore, salbutamol, a beta 2-partial agonist, antagonized the cyclase activation by isoproterenol in AH130 cells. The Kact value of isoproterenol for the activation of adenylate cyclase in AH130 cells was smaller than that in rat liver cells. A comparison of the Ki values of beta-antagonists for the inhibition of isoproterenol-stimulated cyclase activity shows that while the Ki values of propranolol and butoxamine in AH130 cells were similar to those in rat liver cells, a significant difference was observed in the values for beta 1-selective antagonists between AH130 cells and rat liver cells. The Ki values of metoprolol and atenolol for AH130 cells were 137- and 90-fold lower, respectively, than for normal rat liver cells. From these findings, it is strongly suggested that beta-adrenoceptors in rat ascites hepatoma cells including AH130 cells have similar properties to the mammalian beta 1-receptor.

Tumor detection with carbon-11-labelled amino acids

A comparative study of tumor detection with ten 11C-labeled amino acids including four newly synthesized amino acids was carried out to find the most valuable 11C-labeled amino acid for the diagnosis of cancer. 11C-L-methionine showed the highest uptake by the experimental rat hepatoma AH109A (2.7% administered dose/g at 20 min, tumor to blood ratio; 11.4). The second highest uptake was of 11C-aminocyclopentane-carboxylic acid (ACPC). The newly synthesized 11C-DL-methyl-ACPC characteristically showed higher accumulation in tumor than in liver and the tumor to liver ratio reached 3.0 at 60 min after injection. It is suggested that 11C-L-methionine and 11C-DL-methyl-ACPC are useful amino acids for the diagnosis of cancer using positron emission tomography.

Roles of calcium in aggregation of rat ascites hepatoma cells by cell surface-associated adhesive factor.

Our previous studies have shown that a cell surface-associated adhesive factor (AF), separated from rat ascites hepatoma AH136B cells of a differentiated type and highly purified by chromatography, induces not only aggregation of dissociated AH136B cells or rat ascites hepatoma AH109A cells of an undifferentiated type but also adhesiveness characterized by the development of junctional complexes; the AF-induced aggregation of the cells was Ca2+-dependent. Further analysis of the roles of Ca2+ in cell aggregation was performed using AH109A cells (present as single cells in vivo). (1) AF clearly enhanced 45Ca uptake by the cells; (2) calmodulin was isolated from the cells; (3) calmodulin inhibitor, W-7 (N-(6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide), strongly inhibited aggregation of the cells; (4) W-7 also inhibited the clustering or capping of AF-binding sites on the cell surface; (5) binding of 125I-labelled AF to the cells was independent of Ca2+ concentration; (6) binding of 125I-labelled AF to AF-conjugated beads was not observed, independently of the presence of Ca2+. These findings suggest that Ca2+ and Ca2+-activated calmodulin may play a key role in the process of aggregation of the cells by controlling the microfilament components and that Ca2+ may not be involved either in the interactions between AF and its cellular receptor or in linkages of AF molecules.

Affinity of 167Tm-citrate for tumor and liver tissue.

Strong affinity of 167Tm-citrate for tumor tissue was reconfirmed by using Ehrlich tumor. Excellent tumor imaging was obtained with 167Tm-citrate because of its strong tumor affinity and because of the suitable physical characteristics of 167Tm. A large number of 167Tm had accumulated in the connective tissue which contained inflammatory tissue, quite large amounts were found in areas containing viable and necrotic tumor tissue, and small amounts were present in viable tumor tissue. 167Tm was not seen in necrotic tumor tissue. It was concluded that lysosomes did not play a major role in the tumor concentration of 167Tm, but played an important role in the liver concentration of this nuclide. In the case of hepatoma AH109A, it was presumed that lysosomes played a considerably important role in the tumor concentration of 167Tm, hepatoma AH109A possessing some residual features of the liver. 167Tm was bound to acid mucopolysaccharides and transposed by the acid mucopolysaccharides in the tumor tissues and liver. The acid mucopolysaccharides to which 167Tm were bound in tumor and liver, were heparan sulfate, chondroitin sulfate (or keratosulfate) and heparin (or keratosulfate).

A high-affinity [formula omitted] in plasma membranes of rat ascites hepatoma AH109A cells

The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase which possesses a high affinity for free calcium (high-affinity (Ca 2+ + Mg 2+)-ATPase , EC 3.6.1.3) has been detected in rat ascites hepatoma AH109A cell plasma membranes. The high-affinity (Ca 2+ + Mg 2+)-ATPase had an apparent half saturation constant of 77 ± 31 nM for free calcium, a maximum reaction velocity of 9.9 ± 3.5 nmol ATP hydrolyzed/mg protein per min, and a Hill number of 0.8. Maximum activity was obtained at 0.2 μM free calcium. The high-affinity (Ca 2+ + Mg 2+)-ATPase was absolutely dependent on 3–10 mM magnesium and the pH optimum was within physiological range (pH 7.2–7.5). Among the nucleoside trisphosphates tested, ATP was the best substrate, with an apparent K m of 30 μM. The distribution pattern of this enzyme in the subcellular fractions of the ascites hepatoma cell homogenate (as shown by the linear sucrose density gradient ultracentrifugation method) was similar to that of the known plasma membrane marker enzyme alkaline phosphatase (EC 3.1.3.1), indicating that the ATPase was located in the plasma membrane. Various agents, such as K +, Na +, ouabain, KCN, dicyclohexylcarbodiimide and NaN 3, had no significant effect on the activity of high-affinity (Ca 2+ + Mg 2+)-ATPase . Orthovanadate inhibited this enzyme activity with an apparent half-maximal inhibition constant of 40 μM. The high-affinity (Ca 2+ + Mg 2+)-ATPase was neither inhibited by trifluoperazine, a calmodulin-antagonist, nor stimulated by bovine brain calmodulin, whether the plasma membranes were prepared with or without ethylene glycol bis(β- aminoethyl ether)-N,N,N′,N′- tetraacetic acid . Since the kinetic properties of the high-affinity (Ca 2+ + Mg 2+)-ATPase showed a close resemblance to those of erythrocyte plasma membrane (Ca 2+ + Mg 2+)-ATPase , the high-affinity (Ca 2+ + Mg 2+)-ATPase of rat ascites hepatoma cell plasma membrane is proposed to be a calcium-pumping ATPase of these cells.

Subcellular distribution of gallium-67 in tumor and liver

Subcellular distribution of 67Ga was quantitatively determined to evaluate the role of the lysosome in accumulation of 67Ga in malignant tumor tissue and the liver using three different tumor models and the host liver. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity of 67Ga was localized in the supernatant fraction, and only a small amount of radioactivity was localized in the mitochondrial fraction, which contains lysosomes. In the liver, however, most of the radioactivity was concentrated in the mitochondrial fraction. The radioactivity of this fraction increased with time after the administration of 67Ga and reached approximately 50% of total radioactivity within 24 h. In the case of hepatoma AH 109A, radioactivity of the mitochondrial fraction increased with time after administration, and about 30%, of total radioactivity was concentrated in this fraction after 24 h. It is concluded that lysosome does not play a major role in the tumor concentration of 67Ga, although it may play an important role in the liver concentration of 67Ga. In the case of hepatoma AH109A, it is presumed that lysosome plays a considerably important role in the tumor concentration of 67Ga, hepatoma AH 109A possessing some residual features of the liver.

Subcellular distribution of 111In and 169Yb in tumor and liver.

Subcellular distribution of 111In and 169Yb was quantitatively determined to evaluate the role of the lysosome in the accumulation of these nuclides in malignant tumor tissue and in the liver using three different tumor models and the host liver. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity of these nuclides was localized in the supernatant fraction, and only a small amount of radioactivity was localized in the mitochondrial fraction, which contains lysosomes. In the liver, most of the radioactivity was concentrated in the mitochondrial fraction. The radioactivity of this fraction increased with time after the administration of these nuclides and reached approximately 50% of the total radioactivity within 24 h. In the case of hepatoma AH109A, radioactivity of the mitochondrial fraction increased with time after administration, and about 30% of the total radioactivity was concentrated in this fraction after 24 h. It is concluded that the lysosome does not play a major role in the tumor concentration of these nuclides, although it may play an important role in their liver concentration. In the case of hepatoma AH109A, it is presumed that lysosome plays a considerably important role in the tumor concentration of these nuclides, hepatoma AH109A possessing some residual features of the liver.

Effect of attenuated cells on the growth of small implant of Yoshida ascites hepatoma AH109A.

The transplantability of small implants such as 10 cells of Yoshida ascites hepatoma AH109A, which grew lethally only in 2 out of 19 rats when transplanted subcutaneously, was improved by admixed implantation of attenuated tumor cells prepared with mitomycin C, presenting lethal take in 23 out of 27 animals. Similar effect was also obtained by liver homogenate or sonicated tumor cell suspension. Although the rate of tumor "take" of less than 10(3) cells of AH109A inoculated in the subcutis increased by admixing the attenuated cells, growth characteristics of such a small implant of the tumor, once initiated, were not significantly different whether inoculated with or without the attenuated cells. Growth support effect of attenuated cells was discussed in relation with either the size of inoculum or growth ability of tumor cells.

A possible mechanism for island formation by rat ascites hepatoma cells with special reference to the function of aggregation factor at the cell surface

Two tumor cell-aggregation factors of glycoprotein nature, separated from rat ascites hepatoma AH136B cells (forming cell islands in vivo), had different antigenicity; one was not absorbed by immunoadsorbent chromatography with anti-rat serum antibody and the other was. The unabsorbed factor induced aggregation (as shown in the form of simple apposition) of rat ascites hepatoma AH109A cells (present as a free form in vivo) and cell adhesiveness characterized by well-defined tripartite junctional complexes, including intermediate junctions, desmosomes, and tight junctions. In contrast, the absorbed factor from AH136B cells, AH109A cells or normal rat serum aggregated AH109A cells but failed to develop the junctional complexes; only simple apposition was observed. AH109A cells themselves contained the absorbed factor but not the unabsorbed factor. AH136B cells proliferating in the skin developed the junctional complexes, while AH109A cells proliferating in the skin did not from any junctional complexes.

Study of Subcellular Distribution of 67Ga in Tumor and Liver

Subcellular distribution of 67Ga was quantitatively determined to evaluate the role of lysosome in accumulation of 67Ga in malignant tumor tissue and liver. The following animals and transplanted tumors were used: rats implanted with Yoshida sarcoma and hepatoma AH109A; mice implanted with Ehrlich tumor. 67Ga-citrate were injected to the rats intravenously and to the mice intraperitoneally. Ten minutes to 48 hours after the administration of 67Ga-citrate, the animal were sacrificed, and the tumor tissues and liver were excised. Subcellular fractionation of tumor tissues and livers were carried out according to the method of Hogeboom and Schneider. Radioactivity of each fraction was counted by a well type scintillation counter, and protein of each fraction was measured according to Lowry's method. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity was localized in the supernatant fraction, and small amount of radioactivity was localized in the mitochodrial fraction (lysosome contains in this fraction). But in the liver, most of the radioactivity was concentrated in the mitochondrial fraction and the radioactivity of this fraction was increased with the passage of time after administration. Twenty-four hours later, about 50% of total radioactivity was accumulated in this fraction. In the case of hepatoma AH109A, radioactivity of mitochondrial fraction was increased with the passage of time after administration, and about 30% of total activity was concentrated in this fraction at 24 hours after administration. From these results it is concluded that lysosome doses not play an important role in the tumor concentration of 67Ga and lysosome plays an important role in the liver concentration of 67Ga. In the case of hepatoma AH109A it is presumed that lysosome plays considerably important role in the tumor concentration of 67Ga, hepatoma AH109A having some nature of liver.

Hematological alterations in tumor-bearing rats, with reference to pathogenesis of chronic type of disseminated intravascular coagulation syndrome.

Hematological studied were carried out serially in the rats transplanted subcutaneously with Yoshida ascites hepatoma AH-109A. Significant changes were observed in fibrinogen level, fibrinogen degradation products, recalcification time, platelet count, and fragmentation of red blood cells. Formation of thrombi was revealed in the vessels of tumor tissue morphologically from early stage when the tumor grew to a palpable size. Thrombi were formed also in the arterioles of the lungs in the terminal stage. Bleeding tendency was noted in some cases at death. These findings suggested the experimental induction of a type of disseminated intravascular coagulation. The systemic changes of the blood occurring in the terminal stage were preceded by localized intravascular coagulation and fibrinolysis in the tumor in early stage of tumor growth.

The induction of tumour cell adhesiveness and intercellular junctions by a glycoprotein of rat ascites hepatoma cell surface

Rat ascites hepatoma AH109A cells (present as a free form in vivo) can aggregate and then develop well-defined tripartite junctional complexes, including intermediate junctions, desmosomes and focal tight junctions, on incubation with a glycoprotein separated from rat ascites hepatoma AH136B cells (forming cell islnds in vivo). The development of binding structures was strongly inhibited by actinomycin D. AH109A cells or rat ascites hepatoma YS cells (present as a free form in vivo) previously treated with the glycoprotein for 24 h, when inoculated i.p., proliferated as free cells in the ascitic fluid, like the untreated cells. AH109A cells actively proliferating in the skin do not form any junctional complexes. The reason for the failure of island formation by AH109A cells or YS cells in vivo is discussed.

An experimental model for lymphatic metastasis in rats.

An experimental model was described for inducing spontaneous lymph node metastasis, in which tumor cells of the rat ascites hepatoma AH-109A were transplanted into the subcutaneous tissue of the penis, which is rich of lymph plexus. Growth of tumor at the site of transplantation was measured serially and metastasis was recognized by palpation in the inguinal lymph nodes initially, and then in the axillary and lumbar nodes. When animals were sacrificed 12 days after transplantation, these lymph nodes were weighed and high percentage of metastasis was confirmed by histological examination. As an experimental model of lymph node metastasis, the usefulness of this method was discussed.

Effect of sulfated polysaccharides on blood-borne pulmonary metastasis in rats.

The inhibitory effect of sulfated polysaccharides on blood-borne metastasis was examined. As a model of blood-borne metastasis, the ascitic form of hepatoma AH-109A tumor was injected intravenously into Donryu strain rats. Examination of the pulmonary metastatic nodules developed 2 weeks later showed inhibitory effect of the five sulfated polysaccharides tested. Xylan sulfate was the most inhibitory, and exerted its inhibitory effect when the tumor cells were in the pulmonary capillary beds. However, fromthe rapid disappearance of radioactivity from the lungs after injection of 125IUDR-labeled AH-109A cells, tumor cells seemed to be retained in the lungs for only a very short time. Measurement of the anticoagulative and fibrinolytic activities of three sulfated polysaccharides showed that the inhibitory effect of these compounds on blood-borne metastasis was proportional to their anticoagulative and fibrinolytic activities, xylan sulfate showing the highest activities. These results suggest that sulfated polyaccharides may inhibit blood-borne pulmonary metastasis by inhibiting the lodging of tumor cells in the pulmonary capillary beds.

Studies on the mechanisms of invasion in cancer. I. Isolation and purification of a factor chemotactic for cancer cells.

AbstractA substance chemotactic for cancer cells was extracted from the pseudoglobulin fraction of some tumor tissues of animal and human origin. It was highly purified by column chromatography using Sephadex G‐50 and CM‐Sephadex and then by disc electrophoresis; it behaved as a homogenous substance on disc electrophoresis. The substance was a protein with a molecular weight of approximately 70,000. It was thermolabile and had no proteolytic activity. The material was similarly active for rat ascites hepatoma AH109A cells, mouse ascites hepatoma MH134 cells and mouse myeloid leukemia C‐1498 cells, suggesting a common chemotactic action. It was ineffective for polymorphonuclear leukocytes of rats. No such chemotactic activity was demonstrated by the protein fraction from normal skin and muscle.