精氨酸对艾氏腹水癌细胞体外作用的机制探讨

本研究采用小鼠艾氏腹水癌细胞探讨了精氨酸对一些肿瘤细胞体外作用的可能机制。结果表明精氨酸对艾氏腹水癌细胞体外蛋白质合成有显著的抑制作用,其作用受培养介质中一些氨基酸的影响;细胞内游离氨基酸浓度分析结果提示精氨酸的作用可能并不是通过干扰细胞内游离氨基酸池所引起,其具体作用机制尚待进一步实验的揭示。     

Inhibition of the repair of neutron-induced DNA single-strand breaks in experimental tumor cells

Nicotinamide and arabinoside cytosine mixed with hydroxyurea were shown to influence the relative amount of double-stranded DNA in Ehrlich ascites tumor cells in vitro subjected to single irradiation (10-30-52 Gy) and in Guerin's carcinoma in rat lungs exposed to fractionated 6 MeV neutron-radiation (1.25 Gy X 4). The DMF values for Ehrlich ascites tumor were a function of a dose range and the duration of the drugs' effect. Guerin's carcinoma DNA was found to be affected more readily when treated with radiation and drugs than when exposed to neutron radiation alone.     

Membrane fluidity in Ehrlich ascites tumor cells treated with adriamycin

The incubation of Ehrlich ascites tumor cells with adriamycin resulted in an increase in lipid peroxide content and a decrease in membrane fluidity as measured by electron spin resonance using the paramagnetic probe 5-doxylstearic acid. Coincidently, the incorporation of [3H]thymidine into tumor cells was progressively inhibited as the concentration of adriamycin was increased. The results indicate that adriamycin induces changes in the plasma membrane of Ehrlich ascites tumor cells after exposure to a low, but cytotoxic, level of this agent.     

Increases in mRNA levels of glucose transporters types 1 and 3 in Ehrlich ascites tumor cells during tumor development

A common feature of many tumors is an increase in glucose catabolism during tumor growth. We studied the mechanism of this phenomenon by using Ehrlich ascites tumor bearing mice as the animal model. We found that Ehrlich ascites tumor cells possess only glucose transporter 1 (GLUT1) and GLUT3 but no GLUT2, GLUT4, or GLUT5. The mRNA levels of GLUT1 and GLUT3 increased progressively in the tumour during development; however, there were no changes observable in mRNA levels of glucose transporters of all types in brain, liver, and heart of the host mice. These findings suggest that Ehrlich ascites tumor augments its glucose transport mechanism relative to other tissues in response to its unique growth needs. J. Cell. Biochem. 67:131–135, 1997. © 1997 Wiley-Liss, Inc.     

Tumor-Specific Transplantation Resistance in Mice after Treatment of Initial Tumors with Streptococcus thermophilus

Antitumor activity observed by treatment with Streptococcus thermophilus was further investigated. The mice cured from fibrosarcoma by treatment with heat-killed preparation of S. thermophilus, when challenged with fibrosarcoma failed to take up the tumor. However, these cured mice when challenged with sarcoma-180 or Ehrlich ascites carcinoma, did not show significant changes in tumor take and/or survival compared to their respective controls. Similarly, mice cured from sarcoma-180 were challenged with fibrosarcoma, sarcoma-180 or Ehrlich ascites carcinoma. Though there was no change in the mean survival time (MST) of the dying mice regarding sarcoma-180 or Ehrlich ascites carcinoma, there was 50 and 30% increase in the number of mice that showed total regression respectively over controls. However, there was no difference in the growth rate of fibrosarcoma. Similar observations were made with mice cured from Ehrlich ascites carcinoma, challenged with these tumors. These findings thus suggest that the antitumor response was tumor-specific and that tumor-associated antigens may have a role in imparting this specificity. Bacterial treatment non-specifically augmented this primary response.     

Dephosphorylation of the small heat shock protein hsp25 by calcium/calmodulin-dependent (type 2B) protein phosphatase.

The dephosphorylation of the mouse small heat shock protein hsp25 within an extract obtained from Ehrlich ascites tumor cells is inhibited by the calcium chelator EGTA and at concentrations of microcystin-LR which are characteristic for inhibition of calcium/calmodulin-dependent (2B type) protein phosphatases. Furthermore, the dephosphorylation of hsp25 in the cell-free system derived from Ehrlich ascites tumor could be increased specifically by addition of the calcium/calmodulin-dependent (2B type) protein phosphatase calcineurin. Dephosphorylation of the heat shock protein hsp25 is also obtained in an in vitro system containing phosphorylated recombinant hsp25, 1 mM Ca2+, calmodulin, and calcineurin specifying hsp25 as the direct substrate for this enzyme. The expression of two isoforms of the catalytic subunit of the mouse calcium/calmodulin-dependent (2B type) protein phosphatases in Ehrlich ascites tumor cells is demonstrated by polymerase chain reaction using specific oligonucleotide primers to the catalytic and calmodulin-binding domain, respectively. Northern blot analysis using the amplified fragments as probes shows that the mRNA of one isoform of the mouse calcium/calmodulin-dependent protein phosphatase is of medium abundance in EAT cells. These data suggest a calcium/calmodulin-dependent dephosphorylation of the small stress protein in EAT cells also in vivo. Since it is known that heat shock increases the intracellular calcium level and that thermotolerance is influenced by calcium chelators, ionophores, and anti-calmodulin drugs, the changes in the degree of hsp25 phosphorylation induced by thermal stress resulting in an altered thermoresistance could be explained at least partially by the calcium/calmodulin-dependent dephosphorylation through protein phosphatases 2B.     

Variation of cell kinetic parameters in relation to the growth rate of the Ehrlich ascites tumor.

A multicompartmental model of the cell cycle and proliferation kinetics was used to analyse the time-course behavior of the cell cycle time, the growth fraction, and the cell loss rate during Ehrlich ascites tumor growth. The growth rate of Ehrlich ascites tumor cells as the tumor aged was significantly influenced by change in the cell cycle time.     

Changes in the SH-group level and respiration during gamma- and Fe2+-induced lipid peroxidation in Ehrlich ascites carcinoma cells

In experiments on Ehrlich ascites tumor cells it was shown that lipid peroxidation induced by gamma-rays and Fe2+ ions was accompanied by a decrease in the endogenous SH-group content at early times after exposure (during 3-hour incubation). It was also established that no significant changes occurred in the oxygen uptake by Ehrlich ascites tumor cells depending on radiation dose of Fe2+ ion concentration. If cells were pre-kept under hypotonic conditions an additional decrease in cell respiration and SH-group content and activation of lipid peroxidation was noted.     

Effect of nonspecific immune stimulation with BCG and polidin on the Ehrlich ascites tumor growth

Investigations were performed to study: 1) the antitumor effect of BCG pretreatment on the development of Ehrlich ascites tumor in mice; 2) the effect of BCG administration in relation to the period of time before tumor inoculation and the dose levels used, and 3) the antitumor effect of an associated pretreatment of BCG and Polidin on the development of Ehrlich ascites tumor. BCG administered prior to Ehrlich ascites tumor inoculation have a protective effect evidenced by a delay in tumor development, a prolonged survival of the tumor host and, in some cases, even inhibition of tumor growth. The effect of BCG was highly dependent on 1) the dose and the time of administration of BCG and) 2 the combined pretreatment of BCG and Polidin.     

VARIATION OF CELL KINETIC PARAMETERS IN RELATION TO THE GROWTH RATE OF THE EHRLICH ASCITES TUMOR

A multicompartmental model of the cell cycle and proliferation kinetics was used to analyse the time-course behavior of the cell cycle time, the growth fraction, and the cell loss rate during Ehrlich ascites tumor growth. The growth rate of Ehrlich ascites tumor cells as the tumor aged was significantly influenced by change in the cell cycle time.     

Bioflavonoid regulation of ATPase and hexokinase activity in Ehrlich ascites cell mitochondria.

(1) The mitochondrial ATPase (EC 3.6.1.3) Ehrlich ascites cell mitochondria, was inhibited by D-glucose under physiological concentrations of ATP. The generation of ADP by the mitochondrial bound hexokinase, seems to be the reason for the D-glucose inhibitory effect. Reversal of the inhibitory effect of ADP on Ehrlich ascites cell mitochondria ATPase by an ATP-regenerating system was achieved. (2) Dissociation of mitochondrial bound hexokinase from the mitochondria eliminated the inhibitory effect of D-glucose. Rebinding of the hexokinase to the mitochondria regenerated the D-glucose inhibitory effect on Ehrlich ascites cell mitochondria ATPase. (3) Bioflavonoids such as quercetin inhibit the mitochondrial hexokinase activity, but do not change the mitochondrial ATPase activity of isolated Ehrlich ascites tumor cell mitochondria. (4) The inhibitory effect of bioflavonoids on mitochondrial bound hexokinase activity is shown to be dissociable from the ascites tumor cell mitochondria and seems to be associated with regulatory rather than catalitic sites of the enzyme.     

Regulation of protein kinases activity by quercetin in Ehrlich ascites tumor cells

Cyclic AMP-independent protein kinase activities from Ehrlich ascites tumor cells, partially purified by DEAE-cellulose and phosphocellulose chromatography were inhibited by quercetin. The cyclic AMP in the tumor ascites cells and the cyclic AMP-dependent protein kinase activity from this tumor and from bovine and mouse tissues were unaffected by this drug. Since we reported that quercetin elevates cyclic AMP level in Ehrlich ascites tumor cells, this bioflavonoid may have a dual effect on the protein kinae activities in these cells, thus, increasing the cyclic AMP-dependent and decreasing the cyclic AMP-independent protein kinase activities.     

An investigation of the stability of messenger RNAs in cell-free,translational systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells

The stabilities and translation of Ehrlich ascites tumor cell poly(A)-containing mRNA and mengovirus RNA in fractionated cell-free protein synthesizing systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells were studied. During incubation of the systems about 20% of the input RNA is reduced in size and associated with ribosomes engaged in polypeptide synthesis; the remainder is rapidly degraded by RNases. At the end of active translation, both mRNA and nascent proteins are bound to polysomes which are of the same size as those formed during active protein synthesis. The kinetics of protein synthesis closely follow those of RNA hydrolysis. The stabilities of mengovirus RNA and poly(A)-containing mRNA from Ehrlich ascites tumor cells are the same in both systems.     

Localization of 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in the mammalian kidney

The tRNA from Ehrlich ascites tumor cells is deficient in the modified nucleoside Q (queuosine). Continuous infusion of Q base (queuine) to tumor-bearing mice reverses the deficiency of Q in Ehrlich ascites tRNA, and coincidently, causes an inhibition of tumor growth.     

Plasma membrane redox systems in tumor cells

Summary Plasma membrane redox systems in tumor cells are analyzed, their role in proton flux and tumor cell growth is described, and the modulation of their activity by antitumor drugs and growth factors is presented. As an example of the evolution of studies in the characterization of plasma membrane redox systems in tumor cells, we summarized our own results on the model system Ehrlich ascites carcinoma.     

Growth phase-dependent proteins of the Ehrlich ascites tumor analyzed by one- and two-dimensional electrophoresis

The decrease of the growth rate of the Ehrlich ascites tumor is correlated with changes in the de novo synthesis and abundance of several proteins as is shown by electrophoretic methods. The synthesis of a number of these growth-dependent proteins can be induced in vitro by incubation of the tumor cells in the presence of serum.     

Lipid peroxidation and cytotoxicity of Ehrlich ascites tumor cells by ferric nitrilotriacetate

Ferric nitrilotriacetate, which causes in vivo organ injury, induced lipid peroxidation and cell death in Ehrlich ascites tumor cells in vitro. The process was inhibited by butylated hydroxyanisole and enhanced by vitamin C and linolenic acid, indicating a close relationship between cytotoxicity and the lipid peroxidizing ability of Fe3+ NTA. The cytotoxicity was suppressed by glucose and a temperature below 20 degrees C. Lipid peroxidation of Fe3+ NTA-treated cells was greater at 0 degree C than at 37 degrees C, contrary to results with Fe3+ NTA-treated plasma membranes of Ehrlich ascites tumor cell. These results suggested that metabolism and membrane fluidity are important factors in the expression of the Fe3+ NTA-induced cytotoxicity. H2O2 showed a lower cytotoxicity than did Fe3+ NTA but a greater lipid peroxidizing ability. H2O2 appeared to damage the cells less, and was quenched rapidly by cellular metabolism unlike Fe3+ NTA. In transferrin-free medium, Ehrlich ascites tumor cell readily incorporated Fe3+ NTA, and iron uptake was greater than NTA-uptake in Fe3+ NTA-treated cells, suggesting that Ehrlich ascites tumor cell incorporated iron from Fe3+NTA and metabolized it into an inert form such as ferritin.     

The effect of polycations on cell membrane stability and transport processes

Summary The interaction of poly-l-lysines of different molecular weights (PL) with Ehrlich ascites tumor cells was studied experimentally with respect to cell surface binding, cell electrophoresis, cytotoxicity and membrane permeability. Although they decrease the net negative charge of Ehrlich ascites cells similarly at low PL concentrations, low molecular weight PL was less cytotoxic and less damaging to the potassium transport mechanism than was high molecular weight PL. At certain PL concentrations, membrane damage was reversible on reincubation in PL-free media. The amount of bound polylysine as determined with fluorescent labeled polylysine was compared by electrophoresis to the amount of polylysine expressed on the electrokinetic surface. The results indicated that only a small fraction of polylysine bound to Ehrlich ascites tumor cells was electrokinetically detectable. The adsorption of polylysine to Ehrlich ascites tumor cells was not describable by the usual adsorption isotherms. It is suggested that the same number of monomeric lysine units of high and low molecular weight PL are adsorbed at the cell electrokinetic surface, but cytotoxicity is dependent on molecular weight. Although the negative charge of human red blood cells could be reversed at low PL concentrations, no such effect could be observed for ELD (a subline of Ehrlich ascites carcinoma) cells even at high PL concentrations. The relationship of PL binding to the stimulation of macromolecular uptake is discussed.     

1′-Acetoxychavicol acetate-induced cytotoxicity is accompanied by a rapid and drastic modulation of glutathione metabolism

The effect of 1′-acetoxychavicol acetate (ACA), an anticarcinogenic compound naturally obtained from rhizomes and seeds of South East Asia plants, on the intracellular concentration of glutathione and the activities of enzymes related to glutathione metabolism was studied in Ehrlich ascites tumor cells. We showed in a previous study that ACA induced apoptosis in tumor cells and the cell death was reversed by the addition of N-acetlycysteine or glutathione ethylester. Here we found that ACA caused a rapid decrease in glutathione level in less than 10 min after ACA exposure. At the time, glutathione reductase activity was significantly inhibited and gamma-glutamyl cysteine increased by ACA exposure. These results show that ACA caused the decrease in the intracellular GSH levels in Ehrlich ascites tumor cells, suggesting that ACA-induced decrease of the cellular GSH levels can lead to growth arrest of cancer and enhancement of the efficacy other anticancer drugs.     

The modelling of the growth of Ehrlich carcinoma and teratoma T-36 with ascitic fluid and tumor-cell dialysate]

The study of the effect of ascitic fluid and dialysate of Ehrlich ascites tumor cells (M.m. less than 15 kDa) on the growth of Ehrlich carcinoma and teratoma T-36 has shown that both the ascitic fluid and dialysate can protect tumor cells in vivo. The number of animals with tumors increased from 0% in control animals to 60 and 20%, respectively, in experimental ones after transplantation i.m. of 20 x 10(3) Ehrlich tumor cells into mice. Compared to control, ascitic fluid and dialysate of Ehrlich ascites tumor cells increased the rate of tumor growth to 195 and 153%, respectively. It is suggested that this test-system simulates the effect of tumor humoral factors in vivo.