Effects of GTP on cyclic AMP concentrations in intact Ehrlich ascites tumor cells.

Experiments were conducted to test the hypothesis that intracellular concentrations of guanosine triphosphate (GTP) regulate the activity and hormonal sensitivity of adenylate cyclase in intact cells. By appropriate treatments, the GTP concentrations of Ehrlich ascites tumor cells could be varied between 28% and 680% of control values. Cyclic AMP concentrations were measured before and after addition of epinephrine in cells containing this range of GTP concentratins. Basal cyclic AMP concentrations were unaffected by changes in GTP concentrations. In cells containing lowered concentrations of GTP, the cyclic AMP concentration following addition of epinephrine was half that in control cells. Elevation of GTP concentrations above normal and had no effect on cyclic AMP concentrations following epinephrine treatment.     

Regulation of cyclic AMP level and lactic acid production in Ehrlich ascites tumor cells.

1. Quercetin (3.3',4',5,7-pentahydroxy flavone) at the concentration of 10(-4) M, as well as 2-10(-2) M theophylline and 1.5 - 10(-4) M prostaglandin E2 caused maximal rise of cyclic AMP in Ehrlich ascites tumor cells. 2. No additional increase of cyclic AMP level in these cells was found when both quercetin (10(-4) M) and theophylline (2-10(-2) M) were present in the incubation medium, while combination of quercetin (10(-4) M) and prostaglandin E2 (1.5 - 10(-4) M) has a synergistic effect on the level of cyclic AMP. 3. Degradation of cyclic AMP by homogenate of Ehrlich ascites tumor cells was inhibited by both quercetin and theophylline. 4. Quercetin, and to a smaller but significant extent theophylline, inhibited the lactic acid production in Ehrlich ascites tumor cells while prostaglandin E2 did not change the glycolytic rate in these cells. No synergistic inhibitory effect on lactic acid production was found when combinations of quercetin and prostaglandin E2, quercetin and theophylline or prostaglandin E2 and theophylline were tested. 5. Treatment of Ehrlich ascites tumor cells with dextran sulfate abolished the inhibitory effect of quercetin on lactic acid production, while the effect of the bioflavonoid on cyclic AMP levels was not altered.     

Stimulation of virus-induced fusion of Ehrlich ascites tumor cells by 3',5'-cyclic AMP.

HVJ(Sendai virus)-induced fusion of Ehrlich ascites tumor cells was found to be stimulated by treatments which increase the intracellular level of cyclic AMP. This stimulation was optimal at an external concentration of Ca⁺⁺ of about 0.5 mM. During the process of cell fusion, the intracellular concentration of cyclic AMP was increased with a maximum at 2 min after the initiation of the fusion reaction.Evidence is also presented which suggests that the increase of the cyclic nucleotide is a part of control mechanism of HVJ-induced fusion of eukariotic cells. Thus, this cyclic AMP-stimulated process could be one of the step(s) requiring ATP and Ca⁺⁺, both of which are necessary for the overall fusion process of the tumor cells.     

Mechanism of inhibition by cyclic AMP of protein kinase C-triggered respiratory burst in Ehrlich ascites tumour cells

The superoxide anion generation in Ehrlicg ascites tumour (EAT) cells increased more than two-fold in the presence of the tumour promoter, tetradecanoyl phorbol myristate acetate (TPA). Epinephrine and dibutryl cAMP (Bt₂ cAMP) inhibited in a dose-dependent manner, both basal and TPA-triggered superoxide generation in EAT cells. The kinetics of inhibition of superoxide generation showed a maximum inhibition between 30 and 40 min of preincubation with epinephrine or Bt₂ cAMP of EAT cells and coincided with an increase in activity of a phosphoprotein phosphatase. In TPA-treated EAT cells, epinephrine or Bt₂ cAMP increased the phosphatase activity in a dose-dependent manner. In vitro EGTA, EDTA and sodium fluoride inhibited phosphatase activity. Superoxide generation in response to TPA in Triton-permeabilized EAT cells was inhibited by inclusion of the phosphatase in the assay. Taken together, these results clearly suggest that the phosphatase activity in EAT cells develops as a result of protein kinase A (PKA) and protein kinase C (PKC)-mediated phosphorylation of the phosphatase which then mediates dephosphorylation of the PKC-triggered phosphorylation of proteins to inhibit respiratory burst. A cross-talk between PKA and PKC pathways negatively modulates superoxide generation in EAT cells.     

Activation of dolichol-phosphate mannosyltransferase by dibutryl cyclic AMP in rat liver

Radiolabeled mannose incorporation into secretory glycoproteins and immunoprecipitable fibronectin in the incubation media significantly increased (105 and 32 percent respectively) with a corresponding increase in the levels of dolichol-phosphate mannose, dolichol-diphosphate oligosaccharides and dolichol-phosphate mannosyltransferase activity in the rat liver slices when incubated with dibutryl cAMP and ATP. Dibutryl cAMP activated maximally this enzyme in the presence of ATP in the incubation medium. The activation of the enzyme resulted in a two fold increase in Vmax with no apparent change in the Km for GDP mannose. Phosphorylation the rat liver microsomes with catalytic subunit of cAMP dependent protein kinase, resulted in the activation of dolichol-phosphate mannosyltransferase. These results suggest that cAMP modulates protein glycosylation by activating dolicholphosphate mannosyltransferase activity. The activation of this enzyme could be through phosphorylation/dephosphorylation mechanism involving a cAMP dependent protein kinase.     

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.     

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.     

Characteristics of D-leucine uptake by mouse Ehrlich ascites tumor cells.

In a previous paper, we reported that various D-amino acids were taken up several times more effectively than the corresponding L-isomers into several tumors tested in vivo. In order to investigate this further, the in vitro uptake of D-[14C]leucine by Ehrlich ascites tumor cells was investigated in comparison with that of L-[14C]leucine. The distribution ratio, the effects of amino acids and pH, and an approximately linear Lineweaver-Burk plot indicated that D-leucine was transported by an active transport system for L-leucine. Vmax and ku, the first-order rate constant for the unsaturable component, for the uptake of D- and L-leucines decreased with a fall in temperature. The activation energies for Vmax and ku were in the range of 5-10 and 18-21 kcal/mol, respectively. The values for L-leucine were greater than those for D-leucine. Km for D-leucine transport increased with decreasing temperature, whereas Km for L-leucine decreased. This difference suggests that the large alkyl chains of D- and L-leucines bind to different portions of a carrier protein.     

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.     

The transport of chloride in Ehrlich ascites tumor cells.

The steady state transport and distribution of chloride between the intracellular and extracellular phases was investigated when the extracellular chloride concentration was varied by isosmotic replacement with nitrate, bromide and acetate. The results of these experiments show that chloride transport, measured by uptake of 36Cl, is sensitive to the replacement anion. In the presence of nitrate, chloride transport is a linear function of the extracellular chloride concentration. The relationship between chloride transport and extracellular chloride in the presence of bromide is concave upward which suggests that this anion inhibits chloride movement. However, when acetate replaces chloride, the relationship between chloride transport and extracellular chloride is concave downward. The chloride distribution ratio of cells incubated in 145-155mM chloride medium is 0.386 and is not effected by the replacement of chloride with nitrate, bromide or acetate. These findings are consistent with the assertion that chloride transport is composed of two parallel pathways, a diffusional plus a saturating, mediated component. Of the total chloride flux (9.1 mmoles Cl-/kg dry weight per minute) measured in chloride medium (145-155 mM Cl-), the mediated component represents 40% and the diffusional component 60%.     

声化学诱导艾氏腹水瘤细胞凋亡机制初探

本研究采用频率1.43MHz,声强3W/cm2的高频聚焦超声处理艾氏腹水肿瘤细胞,研究超声激活血卟啉诱导艾氏腹水肿瘤细胞凋亡的途径及其与癌细胞内的氧自由基之间的关系。通过细胞免疫组织化学方法检测与癌细胞凋亡相关的Bax,细胞色素c和caspase-3蛋白的动态表达,黄嘌呤氧化酶法检测超氧化物歧化酶活性变化,硫代巴比妥酸法检测膜脂质过氧化物的含量。结果发现超声加血卟啉处理1h,癌细胞胞浆中的三种促凋亡蛋白表达增多,3h时表现为高表达;处理1h的癌细胞,超氧化物歧化酶活性下降,膜脂质过氧化物增多。研究结果表明超声激活血卟啉诱导艾氏腹水肿瘤细胞凋亡可能通过线粒体途径,且与癌细胞受损后产生的氧自由基有关。     

Changes in adenylate energy charge in Ehrlich ascites tumor cells deprived of serum, glucose, or amino acids.

The adenylate energy charge in Ehrlich ascites tumor cells increases when cells are cultivated in serum-limiting medium and decreases when they are incubated in glucose- or amino acid-limited media. Protein synthetic rates decrease in cells deprived of serum, glucose, or amino acids. Supplementation of deprived cells with respective nutrients restores normal protein synthetic rates and adenylate energy charge values. Serum-deprived cells incubated in depleted serum media do not increase their rates of protein synthesis and their adenylate energy charge remains elevated. These results suggest that serum factors regulate protein synthetic rates by mechanisms other than those regulating the availability in cells of glucose or of amino acids. The increased rates of utilization of glucose and of amino acids following the addition of serum are probably due to increased biosynthetic requirements.     

Binucleate cells in the Ehrlich ascites tumor. Autoradiographic labeling

An autoradiographic study was performed on binucleate and mitotic cells in the Ehrlich ascites tumor (EAT) untreated and after treatment with 5-fluorouracil (FU). The number of binucleate cells was greater in the treated tumor than in the controls. It was also observed that the number of labeled mitoses was greater in the Fu-treated tumor. Autoradiographic labeling showed that the cells that proved to be binucleate had previously passed through S-phase; thus, these cells belonged to the proliferative compartment.     

Uptake of radioactive thymidine and cytidine by Ehrlich ascites tumor cells in different stages of growth

Summary In Strong A female mice, the Ehrlich ascites tumor inoculated into the peritoneal cavity grows exponentially for the first 7 days with a doubling time of about 36 hours. The tumor enters then into a late stage during which the number of tumor cells in the peritoneal cavity does not increase. The uptake of intraperitoneally injected thymidine decreases from the exponential to the late stage, mostly because of a decrease in the fraction of cells in DNA synthesis. During the exponential phase, the uptake of thymidine is a function of the amount of radioactive thymidine injected per tumor cell, the utilization decreasing with increasing cell dose. The uptake of intraperitoneally injected cytidine decreases slightly with time after inoculation although the fraction of tumor cells in RNA synthesis remains constant.Dedicated to Professor Friedrich Wassermann with admiration and affection on the occasion of his 80th birthday.This investigation was supported by U.S.P.H.S. Grant CA-05667. The author is a U.S.P.H.S. Research Career Development Awardee.