Evidence for Carrier-Mediated Transport of Monosaccharides in the Ehrlich Ascites Tumor Cell

Evidence for carrier-mediated transport of monosaccharides in the Ehrlich ascites tumor cells was provided through kinetic analysis of data obtained by: (a) studying sugar uptake by dilute cell suspensions with an optical densimetric apparatus, (b) studying sugar uptake by thicker cell suspensions by means of direct chemical analytical methods using packed cell plugs, (c) observing the effects of a competitive inhibitor upon sugar uptake with the chemical analytical method, and (d) measurement of tracer uptake of a high affinity sugar in thick cell suspensions in the absence of net movement. Quantitative application of the data obtained with the above experimental procedures to theoretical model systems derived for both carrier-mediated transport and simple passive diffusion indicated that the results were consonant with predictions for the carrier-mediated transport model, but could not be explained on the basis of uncomplicated diffusion.     

Cell Swelling Activates Phospholipase A2 in Ehrlich Ascites Tumor Cells

Ehrlich ascites tumor cells, loaded with ³H-labeled arachidonic acid and ¹⁴C-labeled stearic acid for two hours, were washed and transferred to either isotonic or hypotonic media containing BSA to scavenge the labeled fatty acids released from the cells. During the first two minutes of hypo-osmotic exposure the rate of ³H-labeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell swelling also causes an increase in the production of ¹⁴C-stearic acid-labeled lysophosphatidylcholine. This indicates that a phospholipase A₂ is activated by cell swelling in the Ehrlich cells. Within the same time frame there is no swelling-induced increase in ¹⁴C-labeled stearic acid release nor in the synthesis of phosphatidyl ¹⁴C-butanol in the presence of ¹⁴C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the swelling induced release of ¹⁴C-labeled arachidonic acid. Taken together these results exclude involvement of phospholipase A₁, C and D in the swelling-induced liberation of arachidonic acid. The swelling-induced release of ³H-labeled arachidonic acid from Ehrlich cells as well as the volume regulatory response are inhibited after preincubation with GDP_βS or with AACOCF₃, an inhibitor of the 85 kDa, cytosolic phospholipase A₂. Based on these results we propose that cell swelling activates a phospholipase A₂—perhaps the cytosolic 85 kDa type—by a partly G-protein coupled process, and that this activation is essential for the subsequent volume regulatory response. Received: 23 July 1996/Revised: 17 June 1997     

Antigenicity and Immunogenicity of Cell Surface Fraction of Ehrlich Ascites Tumor Cells

A membrane fraction corresponding to one-tenth of the dry weight of the whole cell was prepared from Ehrlich ascites tumor cells. Such a fraction retained antigenicity in immune adherence inhibition test using antisera taken from mice hyperimmunized to Ehrlich tumors. The antigenicity was impaired by freezing and thawing, and disappeared after such treatment was repeated more than five times, but was stable to heating up to 60°. The above fraction retained immunogenicity to induce resistance of mice against Ehrlich tumors and retained considerable immunogenicity after drying with acetone-ether.     

Partial Purification of Amino Acid Transport Systems in Ehrlich Ascites Tumor Cell Plasma Membranes

Ehrlich ascites tumor cell plasma membranes were subjected to sequential selective protein extraction to identify protein components associated with amino acid transport. These membranes were extracted with Triton X-100 followed by 2,3-dimethylmaleic anhydride. Approximately 80% of the membrane proteins were extracted by these procedures while the original lipids were largely retained (～70%). The quantity of carbohydrate per milligram protein in the residue increased on extraction, consistent with an enrichment of glycoprotein in the residue.

The residual vesicles display the characteristic properties of Na⁺-coupled amino acid transport. These properties include Na⁺-stimulated uptake and Na⁺-gradient-stimulated uptake leading to an accumulation of the solute against its chemical gradient as well as inhibition of uptake by a competitive amino acid, L-methionine. The extracted vesicles exhibit a peak level of α-aminoisobutyrate uptake six times greater than that expected from equilibration of α-aminoisobutyrate. This accumulation is greater than that obtained with native vesicles, albeit slower. The accelerated exchange diffusion of L-leucine is not measurable in the residual vesicles after dimethylmaleic acid anhydride treatment, although it can be measured after Triton extraction. These results are consistent with the conclusion that the amino acid transport systems “A” (Na⁺-coupled) and “L” (Na⁺-independent) in Ehrlich cells, though having overlapping specificities for amino acids, and distinct physical entities.     

Lipidg Peroxidation in Liver and Ehrlich Ascites Cell Mitochondria

Ehrlich ascites cell mitochondria are highly resistant to lipid peroxidation as compared to liver mitochondria from host animals. Succinate protects mitochondria from peroxidative damage, proteins from crosslinks, enzymes from inactivation of the enzymes and membranes from permeability changes. The sensitivity of Ehrlich ascites cell mitochondrial membranes to lipid peroxidation is significantly increased in sub-mitochondrial particles. Lipid peroxidation in tumour mitochondrial membranes can not be diminished by succinate as effectively as in liver mitochondria. Ascites cell mitochondria seems to be protected very efficiently from peroxidative damage by a glutathione-dependent mechanism.     

Infective Substructures of Sendai Virus from Infected Ehrlich Ascites Tumor Cells

Increase of infectivity for embryonated eggs was observed in Ehrlich ascites tumor cells after intraperitoneal inoculation of Sendai virus into tumor-bearing mice. Virus-induced actinomycin-resistant ribonucleic acid consisting of 14S, 18S, 22S, 35S, and 48S was synthesized, and S antigen was produced in infected cells. The infectivity was suggested to be due to viral ribonucleoprotein for the following reasons: (i) the infectivity was unaffected by V antiserum but was abolished by whole hyperimmune serum, (ii) the infectivity was resistant to ribonuclease, (iii) virus particles were found neither in cells nor on red blood cell stroma treated with cellular extracts, (iv) structures similar to Sendai virus ribonucleoprotein with a maximal length of 10,500 A were observed in cellular extracts.     

The Nucleolus and Parachromatin of the Ascites Tumor Cell

1. A method is described for distinguishing the ribonucleoproteins of the nucleolus and parachromatin of ascitic tumor cells of the mouse. 2. In these cells the transfer of ribonucleoprotein from the nucleus to the cytoplasm can occur in two ways. (a) At the end of prophase the nucleolus separates from the chromosomes and nucleolar fragments are released into the cytoplasm. (b) During prophase the parachromatin is aggregated to form parachromatin bodies which are discharged into the cytoplasm, where they can be detected during metaphase, anaphase, and telophase. 3. A metachromatic form of RNA is demonstrable, and may be synthesized, in close relation to the chromosomes during prophase, metaphase, and anaphase. During telophase the distribution of metachromatic RNA changes, the chromatin loses its metachromasia, and intranuclear metachromatic parachromatin becomes evident.     

Reconstitution of Neutral Amino Acid Transport Systems from Ehrlich Ascites Tumor Cells

Amino acid transport systems for alanine and leucine were reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1mM dithiothreitol, and 0.5 mM EDTA a mixture that solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue-staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valino-mycin-induced potassium diffusion seemed to stimulate alanine uptake further.     

Interferon Treatment of Ehrlich Ascites Tumor Cells: Effects on Exogenous mRNA Translation and tRNA Inactivation in the Cell Extract

We reported earlier that in cell extracts that were prepared from interferon-treated Ehrlich ascites tumor cells and preincubated and passed through Sephadex G-25 (S30_INT), the translation of exogenous mRNA (viral and host) was impaired and the impairment could be overcome to a large extent by adding a crude tRNA preparation from Ehrlich ascites tumor cells but not from Escherichia coli. We find now that the rate of inactivation of some tRNA's (especially those specific for leucine, lysine, and serine) but not those of many others is faster in S30_INT than in corresponding extracts from control cells. This increased rate of tRNA inactivation may perhaps account for the need for added RNA to overcome at least partially the impairment of translation in S30_INT. The relationship of the increased rate of tRNA inactivation to the antiviral effect of interferon is unclear. So far no significant difference has been detected in the amount of tRNA needed to overcome the impairment of encephalomyocarditis virus RNA translation in S30_INT between tRNA from interferon-treated cells and tRNA from control cells. Furthermore, no difference was found in the rate of inactivation in S30_INT between leucine-specific tRNA's from interferon-treated and from control cells. tRNA's specific for leucine and lysine were not inactivated (unless very slowly) during incubation under our conditions in an extract from interferon-treated (or from control) cells unless the extract had been passed through Sephadex G-25 or dialyzed. The translation of exogenous mRNA was, however, impaired in an extract from interferon-treated cells that had not been passed through Sephadex G-25. This impairment was apparently not overcome by added tRNA.     

Subcutaneous Growth of Staphylococcus aureus Concomitantly Inoculated with Ehrlich Ascites Tumor Cells

Intratumoral growth of Staphylococcus aureus Cowan I-derived AP332 was examined by subcutaneous inoculation of cocci in doses ranging from 18 to 1.8 × 10⁵ CFU with Ehrlich ascites tumor cells. Inoculation of 18 CFU AP332 resulted in staphylococcal growth in one of five mice, and the proportion of mice established intratumoral infection increased with the initial inocula. Six other strains of S. aureus also grew in the tumor tissue, and none of the three strains of coagulase-negative staphylococci grew at all. Ethanol-killed tumor cells did not promote staphylococcal growth as vigorously as the live tumor cells, especially when the initial inoculum of AP332 was smaller than 10⁴ CFU.     

Reconstitution of Neutral Amino Acid Transport System from Ehrlich Ascites Tumor Cells

Amino acid transport systems for alanine and leucine have been reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1 mM dithiothreitol, 0.5 mM EDTA, a mixture which solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valinomycin-induced potassium diffusion seemed to stimulate alanine uptake further.     

Ehrlich Ascites Tumor Preservation for Fifteen Years—a Simple Method

The Ehrlich ascites tumor was preserved for 15 years by adding to it an equal volume of 40% glycerol and placing the preparation directly into a freezer at -60 C.     

Some Characteristics of DNA Synthesis and the Mitotic Cycle in Ehrlich Ascites Tumor Cells

In vivo studies of Ehrlich ascites tumor cells during the first 5 days of growth in peritoneal cavities of mice consisted of the following: 1. Determination of growth curves by direct enumeration of cells. 2. Estimation of the duration of each phase of the mitotic cycle based on incidence of cells in different phases. 3. Radioautographic studies to determine the proportion of cells in different phases of the mitotic cycle that incorporate tritiated thymidine during a single brief exposure to this precursor of DNA. 4. Estimation of the rate of incorporation of tritiated thymidine at different times during the period of DNA synthesis by comparison of mean grain counts over nuclei in radioautographs at different times following exposure to tritiated thymidine. The assumptions underlying these experiments and our observations concerning the duration of the period of DNA synthesis and its relation to the mitotic cycle are discussed. It is concluded that DNA synthesis is continuous, occupying a period of 8.5 hours during the interphase and that the average rate of synthesis is approximately constant.     

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。     

Role of LTD4 in the Regulatory Volume Decrease Response in Ehrlich Ascites Tumor Cells

Stimulation with leukotriene D₄ (LTD₄) (3–100 nm) induces a transient increase in the free intracellular Ca²⁺ concentration ([Ca²⁺] _i ) in Ehrlich ascites tumor cells. The LTD₄-induced increase in [Ca²⁺] _i is, however, significantly reduced in Ca²⁺-free medium (2 mm EGTA), and under these conditions stimulation with a low LTD₄ concentration (3 nm) does not result in any detectable increase in [Ca²⁺] _i . Addition of LTD₄ (3–100 nm) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD₄-induced (100 nm) acceleration of the RVD response is also seen in Ca²⁺-free medium and also at 3 nm LTD₄, indicating that LTD₄ can open K⁺- and Cl⁻-channels without any detectable increase in [Ca²⁺] _i . Buffering cellular Ca²⁺ with BAPTA almost completely blocks the LTD₄-induced (100 nm) acceleration of the RVD response. Thus, the reduced [Ca²⁺] _i level after BAPTA-loading or buffering of [Ca²⁺] _i seems to inhibit the LTD₄-induced stimulation of the RVD response even though the LTD₄-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca²⁺] _i . The LTD₄ receptor antagonist L649,923 (1 μm) completely blocks the LTD₄-induced increase in [Ca²⁺] _i and inhibits the RVD response as well as the LTD₄-induced acceleration of the RVD response. When the LTD₄ receptor is desensitized by preincubation with 100 nm LTD₄, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD₄ plays a role in the activation of the RVD response. LTD₄ seems to activate K⁺ and Cl⁻ channels via stimulation of a LTD₄ receptor with no need for a detectable increase in [Ca²⁺] _i . Received: 25 September 1995/Revised: 25 January 1996     

Enhanced Antitumor Immunity Contributes to the Radio-Sensitization of Ehrlich Ascites Tumor by the Glycolytic Inhibitor 2-Deoxy-D-Glucose in Mice

Two-deoxy-D-glucose (2-DG), an inhibitor of glycolysis differentially enhances the radiation and chemotherapeutic drug induced cell death in cancer cells in vitro, while the local tumor control (tumor regression) following systemic administration of 2-DG and focal irradiation of the tumor results in both complete (cure) and partial response in a fraction of the tumor bearing mice. In the present studies, we investigated the effects of systemically administered 2-DG and focal irradiation of the tumor on the immune system in Ehrlich ascites tumor (EAT) bearing Strain “A” mice. Markers of different immune cells were analyzed by immune-flow cytometry and secretary cytokines by ELISA, besides monitoring tumor growth. Increase in the expression of innate (NK and monocytes) and adaptive CD4⁺cells, and a decrease in B cells (CD19) have been observed after the combined treatment, suggestive of activation of anti-tumor immune response. Interestingly, immature dendritic cells were found to be down regulated, while their functional markers CD86 and MHC II were up regulated in the remaining dendritic cells following the combination treatment. Similarly, decrease in the CD4⁺ naïve cells with concomitant increase in activated CD4⁺ cells corroborated the immune activation. Further, a shift from Th2 and Th17 to Th1 besides a decrease in inflammatory cytokines was also observed in the animals showing complete response (cure; tumor free survival). This shift was also complimented by respective antibody class switching followed by the combined treatment. The immune activation or alteration in the homeostasis favoring antitumor immune response may be due to depletion in T regulatory cells (CD4⁺CD25⁺FoxP3⁺). Altogether, these results suggest that early differential immune activation is responsible for the heterogenous response to the combined treatment. Taken together, these studies for the first time provided insight into the additional mechanisms underlying radio-sensitization by 2-DG in vivo by unraveling its potential as an immune-modulator besides direct effects on the tumor.     

Leukotriene D4-induced Ca2+ Mobilization in Ehrlich Ascites Tumor Cells

Stimulation of Ehrlich ascites tumor cells with leukotriene D₄ (LTD₄) within the concentration range 1–100 nm leads to a concentration-dependent, transient increase in the intracellular, free Ca²⁺ concentration, [Ca²⁺] _i . The Ca²⁺ peak time, i.e., the time between addition of LTD₄ and the highest measured [Ca²⁺] _i value, is in the range 0.20 to 0.21 min in ten out of fourteen independent experiments. After addition of a saturating concentration of LTD₄ (100 nm), the highest measured increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-containing medium is 260 ± 14 nm and the EC₅₀ value for LTD₄-induced Ca²⁺ mobilization is estimated at 10 nm. Neither the peptido-leukotrienes LTC₄ and LTE₄ nor LTB₄ are able to mimic or block the LTD₄-induced Ca²⁺ mobilization, hence the receptor is specific for LTD₄. Removal of Ca²⁺ from the experimental buffer significantly reduces the size of the LTD₄-induced increase in [Ca²⁺] _i . Furthermore, depletion of the intracellular Ins(1,4,5)P₃-sensitive Ca²⁺ stores by addition of the ER-Ca²⁺-ATPase inhibitor thapsigargin also reduces the size of the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-containing medium, and completely abolishes the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-free medium containing EGTA. Thus, the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells involves an influx of Ca²⁺ from the extracellular compartment as well as a release of Ca²⁺ from intracellular Ins(1,4,5)P₃-sensitive stores. The Ca²⁺ peak times for the LTD₄-induced Ca²⁺ influx and for the LTD₄-induced Ca²⁺ release are recorded in the time range 0.20 to 0.21 min in four out of five experiments and in the time range 0.34 to 0.35 min in six out of eight experiments, respectively. Stimulation with LTD₄ also induces a transient increase in Ins(1,4,5)P₃ generation in the Ehrlich cells, and the Ins(1,4,5)P₃ peak time is recorded in the time range 0.27 to 0.30 min. Thus, the Ins(1,4,5)P₃ content seems to increase before the LTD₄-induced Ca²⁺ release from the intracellular stores but after the LTD₄-induced Ca²⁺ influx. Inhibition of phospholipase C by preincubation with U73122 abolishes the LTD₄-induced increase in Ins(1,4,5)P₃ as well as the LTD₄-induced increase in [Ca²⁺] _i , indicating that a U73122-sensitive phospholipase C is involved in the LTD₄-induced Ca²⁺ mobilization in Ehrlich cells. The LTD₄-induced Ca²⁺ influx is insensitive to verapamil, gadolinium and SK&F 96365, suggesting that the LTD₄-activated Ca²⁺ channel in Ehrlich cells is neither voltage gated nor stretch activated and most probably not receptor operated. In conclusion, LTD₄ acts in the Ehrlich cells via a specific receptor for LTD₄, which upon stimulation initiates an influx of Ca²⁺, through yet unidentified Ca²⁺ channels, and an activation of a U73122-sensitive phospholipase C, Ins(1,4,5)P₃ formation and finally release of Ca²⁺ from the intracellular Ins(1,4,5)P₃-sensitive stores. Received: 9 February 1996/Revised: 15 August 1996     

Separate Swelling- and Ca2+-activated Anion Currents in Ehrlich Ascites Tumor Cells

A Ca²⁺-activated (I _Cl,Ca) and a swelling-activated anion current (I _Cl,vol) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying currents were activated by an increase in the free intracellular calcium concentration ([Ca²⁺] _i ), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular Cl⁻ concentration. I _Cl,Ca current density increased with increasing [Ca²⁺] _i , and this current was abolished by lowering [Ca²⁺] _i to <1 nm using 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA). In contrast, activation of I _Cl,vol did not require an increase in [Ca²⁺] _i . The kinetics of I _Cl,Ca and I _Cl,vol were different: at depolarized potentials, I _Cl,Ca as activated in a [Ca²⁺] _i - and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, I _Cl,vol exhibited time- and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials. The deactivation of I _Cl,vol was dependent on the extracellular Mg²⁺ concentration. The anion permeability sequence for both currents was I ⁻ > Cl⁻ > gluconate. I _Cl,Ca was inhibited by niflumic acid (100 μm), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 μm) and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS, 100 μm), niflumic acid being the most potent inhibitor. In contrast, I _Cl,vol was unaffected by niflumic acid (100 μm), but abolished by tamoxifen (10 μm). Thus, in Ehrlich cells, separate chloride currents, I _Cl,Ca and I _Cl,vol, are activated by an increase in [Ca²⁺] _i and by cell swelling, respectively. Received: 12 November 1997/Revised: 5 February 1998     

Immunological Studies on Antisera of Mice Immunized with Dried or DEPC-treated Ehrlich Ascites Tumor Cells

Intracellular arylsulfatases from Klebsiella aerogenes W70 cells grown in methionine medium (M enzyme) and inorganic sulfate medium containing tyramine (T enzyme) were purified respectively by fractionation with (NH₄)₂SO₄, followed by successive chromatographies on DEAE cellulose, hydroxylapatite, Sephadex G-100 and DEAE Sephadex A-25. On polyacrylamide gel electrophoresis, the two enzymes gave single bands with the same mobilities. Molecular weights of both, determined by SDS gel electrophoresis and by Sephadex G-100 chromatography, were 47,000 and 45,000, respectively. Their activities were maximal at pH 7.5. The affinities of the enzymes (M and T enzymes) for their substrate (Km) and the maximum velocity of hydrolysis (V_max) were enhanced by addition of electron withdrawing substituents. The enzymes were inhibited by inorganic phosphate, cyanide, hydroxylamine and tyramine. The inhibition by tyramine was competitive (Ki = 1.0 × 10^?4 m). These results show that the two enzymes were identical. This was confirmed by the fact that mutant strains, which were unable to synthesize arylsulfatase when grown with methionine, could also not synthesize the enzyme when grown with tyramine.