Effect of hyperthermia on growth of Ehrlich ascites tumor cells

Hyperthermic treatment at 43 degrees C suppressed the growth of Ehrlich ascites tumor (EAT) cells in vitro. Incubation of EAT cells at 43 degrees C for as little as 1.5 h totally abolished the transplantability of the tumor. At the same time, the rate of cellular glucose uptake, the density of glucose transporter on the cells as well as the extent of thymidine, uridine and leucine incorporation were significantly reduced.     

Interaction andin vivo growth inhibition of Ehrlich ascites tumor cells by jacalin

Jacalin has been found to agglutinate Ehrlich ascites cells. The agglutination was inhibited by α-glycosides of D-Gal and β -D-Gal(1 → 3)-D-GalNAc suggesting that the lectin-ascites interaction was carbohydrate-specific. There was 21.8% inhibition of tumour (ascites) cell growthin vivo in mice administered 50μg of jacalin by injection for 6 days following intraperitoneal injection of ascites cells. Administration of 100, 150 and 200μg jacalin resulted in 40.2, 57.5 and 83% inhibition respectively. Thein vivo inhibition of tumour cells growth by jacalin was due to its preferential binding with D-Gal-α -(1 → 6) present as terminal residues in the glycoprotein on tumour cell surface.     

Uridine, but not cytidine can sustain growth of Ehrlich ascites tumor cells in glucose-deprived medium with altered proliferation kinetics

Cell cycle kinetics and energy metabolism of Ehrlich ascites tumor cells grown in glucose-deprived medium supplemented with uridine, were investigated in order to extend our knowledge about the significance of the metabolic conversion of glucose for cell cycle progression of these cells. Viability (dye exclusion test) of uridine supplemented cells was not affected, although growth was reduced to 50 to 60% of the controls. Uridine did not significantly impair growth of controls in standard medium up to 20 mM. Studies on cell cycle progression using flow cytometry (BrdU-H33258 technique) revealed an accumulation of cells in G2M phase, which can be explained by a delay in the passage of G2M-compartment of about 12 +/- 2 h. After a 24 h culture period, 60% of the cell populations were found in G2M (30% in control cultures). This fraction increased to about 70% in the following passage. Protein and DNA synthesis corresponded to the proliferation rate. Oxygen uptake was increased by about 50%, glutamine consumption by 30%, lactate production was reduced to below 10% of the controls. The ATP/ADP concentration ratio was found in a physiological range of 4 to 6. It was calculated that cells grown in standard medium produced 60% of ATP via oxidative pathways and 40% via glycolysis; however, in glucose-free, uridine-supplemented medium the values are more than 90% and less than 10%. No significant differences in total ATP production were observed. Growth of the cells in glucose-deprived medium could not be sustained by cytidine. All our data substantiate the present concept that glycolytic ATP-production is not essential for maintenance of viability and growth of these cells.     

Mannose toxicity in Ehrlich ascites tumor cells

Mannosephosphate isomerase (MPI) showed a higher activity than hexokinase (HKM) in its ability to phosphorylate mannose in the spleen, thymus, brain, liver, striated muscles, kidneys, and testes from BALB/c mice. This led to a HKM/MPI ratio of less than 1 in all the organs and tissues mentioned. In contrast, Ehrlich ascites tumor cells obtained from the peritoneum of BALB/c mice had low MPI activity (half of the HKM activity and, therefore, a ratio of 2). Mannose, which is nontoxic to nontumor cells at a concentration of 0.1 M, induced marked in vitro mortality of the tumor cells. Incubation of Ehrlich ascites tumor cells with mannose resulted in a high accumulation of mannose-6-phosphate and a marked depletion of ATP which did not appear when the cells were incubated with glucose. These facts may explain the selective mortality caused by mannose in the tumor cells studied.     

声化学激活血卟啉诱导艾氏腹水肿瘤细胞凋亡

本实验采用频率为2．0MHz，声强分别为1．0w／cm^2、1．5w／cm^2、2．0w／cm^2等不同参数，研究超声激活血卟啉对艾氏腹水肿瘤细胞的杀伤作用和诱导肿瘤细胞凋亡现象。通过扫描电镜、透射电镜以及荧光显微镜观察受损后细胞形态结构的变化，主要表现为细胞微绒毛的减少，胞膜结构和通透性的改变，细胞器的受损以及核物质的分解、丢失；同时发现处理后的肿瘤细胞有核物质凝集、趋边排列以及凋亡小体的形成等细胞凋亡特征。研究中首次发现声化学激活血卟啉在对艾氏腹水肿瘤细胞杀伤的同时，也能诱导艾氏腹水肿瘤细胞发生凋亡，提示在声动力疗法中并存着对癌细胞的直接杀伤和通过诱导癌细胞凋亡的两种抗癌途径。     

Permeabilization of Ehrlich ascites tumor cells by human serum

Human serum rapidly permeabilized Ehrlich ascites tumor cells to inorganic cations such as Rb⁺ and Ca²⁺; serum from several other species showed little or no activity. The effect of human serum was not reversed by washing the cells. Human serum, deficient in specific complement proteins, had no activity, but was reactivated by the addition of the missing complement component. Since Ca²⁺ was not required for the permeabilization, the alternative pathway of complement activation was implicated. Human serum deficient in Factor B of the alternative pathway was ineffective, but permeabilizing activity was restored by addition of Factor B. Rb⁺ uptake of several other cells was not inhibited by human serum. We conclude that an interaction between human complement and Ehrlich ascites tumor cells is responsible for the membrane lesion observed.     

Uptake of ferrous iron histidinate, a promoter of lipid peroxidation, by Ehrlich ascites tumor cells

The kinetics of the uptake of Fe(II)-histidinate, a known promoter of lipid peroxidation, into Ehrlich ascites tumor (EAT) cells and the intracellular binding of iron were studied in vitro. EAT cells (27.10(6)/ml) were incubated in Hanks' balanced salts solution at 37 degrees C for various time intervals in the presence of FeSO4 (1 mM) and L-histidine (10 mM). Total iron was determined by the 1,10-phenanthroline/ascorbate method and ferric iron by reaction with 5-sulfosalicylic acid; the difference was ascribed to ferrous iron. Total iron decreased rapidly in the medium (242 nmol within the first 10 min), and a corresponding increase of total iron (saturation value 376 nmol after 60 min) was determined within the cells, after the cellular proteins had been solubilized with 6 M urea. In the absence of EAT cells, Fe(II)-histidinate was readily oxidized to Fe(III)-histidinate by oxygen, but this reaction was strongly retarded by the tumor cells. The uptake of iron histidinate occurred in the oxidized state, while an uptake of ferrous iron could not be proven unambiguously. When EAT cells were saturated with iron, it was found that 93% of intracellular iron was bound to water-insoluble proteins and 7% was associated with soluble proteins, while no unbound iron was detectable by the method used. It was concluded that, despite the high uptake of total iron, only a very small portion of the intracellular iron was available as a redox catalyst for lipid peroxidation.     

Nucleosidediphosphate kinase from Ehrlich ascites tumor cells

A phosphate-incorporating protein has been highly purified from the cytosol of Ehrlich ascites tumor cells (EAT cells). The nitrocellulose membrane method was used to follow the progress of the purification by quantitation of the [32P]phosphorylated form of the protein. The purified protein was identified as an NDP-kinase since it exhibited NDP-kinase activity and had enzyme characteristics in common with other NDP-kinases from various mammalian cells. The purified NDP-kinase was found to have a molecular weight of approximately 76,000 daltons. Moreover, the enzyme appears to consist of two distinct polypeptides (18,000 and 20,000 daltons). This enzyme contained 19 amino acids, with high levels of glycine (9.8%) and lysine (9.0%). The enzyme rapidly formed a [32P]phosphoenzyme when incubated with [gamma-32P]ATP in the presence of Mg2+ (1 mM) at the optimum pH of 7.5 even at low temperature (below 4 degrees C). This phosphoenzyme is an enzyme-bound, high-energy-phosphate intermediate, because ATP was formed from it on incubation with ADP in the presence of Mg2+ (1 mM). This finding suggests that the phosphoenzyme functions as an intermediate in NDP-kinase action.     

Cell volume regulation in Ehrlich ascites tumor cells

Ehrlich cells subjected to anisoosmolar media show very rapid volume changes. In hypertonic media they shrink. In hypotonic media they swell but the rapid initial swelling is followed by a regulatory shrinkage lasting ca. 30 minutes. Cells suspended in media with identical ionic concentrations but different total osmolarity (adjusted by sucrose) were compared. These studies revealed that swollen cells adjust their volume by decreasing the amount of intracellular K⁺ and ninhydrin positive substances. Intracellular Na⁺ and ATP concentrations were unchanged. Accordingly ⁴²K⁺ flux analysis showed that the (passive) cell membrane permeability for K⁺ is increased to a minor degree and the Na⁺ permeability unaffected. The increased K⁺ permeability could not be correlated to an increase in ⁴⁵Ca²⁺ influx.     

Thymidylate synthetase and thymidine kinase activities and methotrexate cytotoxicity during growth of L1210 and Ehrlich ascites tumor

The specific activities of TS and TK determined in the cytosols of Ehrlich and L1210 ascites tumor cells changed significantly during their logarithmic growth. In both tumors, the highest activity of TK was in early log phase while that of TS occurred in late logarithmic growth. The activity curves of TS were practically the same and those of TK were similar in the two tumors; however, the absolute values of the latter were different. TK/TS activity ratios in the early and late log growth of Ehrlich tumor were 20 and 2, in those of L1210 tumor 4 and 0.15 respectively. MTX cytotoxicity was considerable throughout the logarithmic growth of L1210 tumor, significantly higher values were observed however when TS activity increased. The variable degree of MTX cytotoxicity indicated an intracellular manifestation of the changes in TS activity determined in vitro. The different patterns for the specific activities of TS and TK, as well as, the parallel changes of TS activity and MTX cytotoxicity during tumor growth may be useful information for the antimetabolite research carried out with experimental tumors.