Inhibition of protein synthesis by antagonists of calmodulin in Ehrlich ascites tumor cells

Several recent publications indicate that Ca2+ is required for protein synthesis in mammalian cells, including the Ehrlich ascites tumor cell. The present communication examines whether the effects of Ca2+ might be mediated through calmodulin or a related protein. Four calmodulin antagonists belonging to different chemical categories were used to provide evidence of calmodulin involvement. Three of the antagonists inhibited protein synthesis in intact cells; 50% inhibitory concentrations were 10 microM calmidazolium, 12 microM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and 17.5 microM trifluoperazine (TFP). Initiation was preferentially inhibited as indicated by an increase in the 80S monomers accompanied by a significant disaggregation of polyribosomes. All the antagonists also inhibited protein synthesis initiation in the cell-free protein-synthesizing system; 50% inhibitory concentrations for compound 48/80, calmidazolium, TFP, and W7 were 10 microM, 125 microM, 300 microM and 500 microM, respectively. A weak analogue of W7 inhibited only 20% at 1000 microM. Inhibition in the cell-free system was reversed by the addition of exogenous calmodulin in all four cases. The levels of 43S complexes were significantly elevated with all four antagonists, indicating a block in the utilization of 43S complexes. The similarity of the effects of four distinct classes of antagonists and their ready reversal by exogenous calmodulin leads us to suggest that there may be a role for calmodulin or a very similar calcium-binding protein in protein synthesis.     

Inhibition of lactate transport and glycolysis in Ehrlich ascites tumor cells by bioflavonoids.

Bioflavonoids are potent inhibitors of lactate transport in Ehrlich ascites tumor cells. The most effective bioflavonoids have four to five hydroxyl groups. Sugar substitution at carbon three, or reduction of the double bond between carbons two and three, decreases their inhibitory activity. Quercetin, the most extensively studied of these compounds, inhibits lactate efflux by 50% at 0.1 micrograms/mg of protein. On addition of quercetin to glycolyzing Ehrlich ascites tumor cells, lactate accumulates inside the cell and the intracellular pH drops. Total lactate production is also inhibited. Nigericin prevents the internal acidification that occurs in the presence of quercetin and also reduces the inhibition of glycolysis. Thus, it appears that inhibition of lactate efflux can affect glycolysis through a lowering of the intracellular pH. The inhibitory effect of quercetin on glycolysis can be explained by its effect on lactate efflux and its previously reported effect on the Na+--K+ ATPase [Suolinna, E.--M., et al. (1974) J. Natl. Cancer Inst. 53, 1515].     

Interferon induction of polyamine-dependent protein kinase activity in Ehrlich ascites tumor cells

Treatment of Ehrlich ascites tumor cell cultures $\text{in}\text{vitro}$ with interferon induces a protein kinase activity that is activated by the polyamines, spermidine and spermine. Putrescine antagonizes the activation. The protein kinase yields a phosphorylated endogenous polypeptide of M_r 68,000–70,000. The polyamine-dependent protein kinase activity cofractionates with a double-stranded RNA-dependent protein kinase activity during affinity chromatography on poly (I) ·poly (C) - agarose or by chromatography on phosphocellulose. The double-stranded RNA-dependent protein kinase also phosphorylates an endogenous polypeptide of M_r 68,000–70,000. Unsuccessful attempts to discriminate between these two protein kinase activities on the bases of their respective capacities to be activated by either double-stranded RNA or spermidine/spermine, suggest that a single protein kinase enzyme may be activated by these strikingly dissimilar modifiers.     

Leukotriene-D4 induced cell shrinkage in Ehrlich ascites tumor cells

Summary The nature of the leukotriene-D₄ (LTD₄) induced cell shrinkage in Ehrlich ascites tumor cells has been investigated. LTD₄ treatment of Ehrlich cells induces net loss of cellular KCl and cell shrinkage independent of the initial cell volume. LTD₄ also produces water loss and reduction in cell volume when all extracellular and all intracellular Cl has been replaced by NO₃. On the other hand, LTD₄ fails to produce any significant changes in cell volume in the presence of the K-channel blocker quinine, suggesting that LTD₄ in Ehrlich cells induces Cl-independent K loss through the Ca²⁺-dependent K channels. However, the effect of physiological doses of LTD₄ on cell volume seems not to be as potent in Cl-free, NO₃ cells when compared to Cl-containing cells, indicating that LTD₄ in Ehrlich cells also provokes Cl-dependent K loss. LTD₄ seems not to produce K loss through an electroneutral K⁺/H⁺ exchange system. LTD₄ still produces Cl-independent K loss and cell shrinkage in the presence of the anticalmodulin drug pimozide but not in the presence of the LTD₄ receptor antagonist L-649,923 or the 5-lipoxygenase inhibitor NDGA. Pretreatment of the cells with pertussis toxin, which inactivates inhibitory guanine nucleotide binding proteins (G-proteins), leads to partial inhibition of the LTD₄-induced shrinkage. It is suggested that the LTD₄-induced activation of K and Cl transporting systems in Ehrlich ascites tumor cells is mediated via a G-protein coupled receptor and that LTD₄ might exert its effect through another lipoxygenase product. The Ca²⁺-calmodulin complex is not involved in the LTD₄-induced activation of K and Cl transporting systems.     

Inhibition of virus-induced fusion of Ehrlich ascites tumor cells by cytochalasin B and D.

Cytochalasin B and D were found to inhibit HVJ (Sendai virus)-induced fusion of Ehrlich ascites tumor cells. Nearly complete inhibition was attained by 4 uM (2 μg/ml) cytochalasin D, whereas cytochalasin B was a less effective inhibitor. The inhibition was largely reversible. Since the transport of 2-deoxy-glucose into the tumor cells was not affected by cytochalasin D (though inhibited by cytochalasin B), the observed inhibition was not related to the effect of the drugs on sugar transport. Instead, it was suggested that the inhibition was due to the action of the drugs on microfilaments. The requirement of ATP for the cell fusion could be explained at least partly by the involvement of microfilaments in the cell fusion process.     

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。     

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.     

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.     

Reversibility of ouabain induced inhibition of cell division and cation transport in Ehrlich ascites cells

Ouabain induced inhibition of cation transport and cell division in Ehrlich mouse ascites tumor cells is reversible, suggesting that this agent does not bind irreversibly to its site of action.