Absence of anthracycline-induced degradation of nuclear DNA in Ehrlich ascites tumour cells

The in vivo effects of anthracycline antibiotics on the integrity of Ehrlich ascites tumour cell DNA have been studied by sedimentation analysis of nuclear structures containing superhelical DNA in neutral sucrose gradients. These fast-sedimenting protein-DNA complexes may be released by gently lysing cells in solution containing non-ionic detergents and high NaCl concentrations (1.95 M). The supercoiled structure of DNA in these protein-DNA complexes is suggested by the characteristic sedimentation in the presence of intercalating agents. Apparently, no DNA damage could be detected in Ehrlich cells from 7-day-old tumours within 3 h after various doses of daunomycin (0.5–10 mg/kg of body wt.) were administered i.p. to mice. Sedimentation anomalies could not be observed even 15 or 30 h after administration of therapeutic doses of daunomycin or adriamycin. In contrast, at 30 min after administration to mice, therapeutic doses of bleomycin (2–8 mg/kg) caused extensive fragmentation of tumour cell DNA, which could be monitored as slowly sedimenting DNA structures (compared with the control). Similarly, DNA damage could be induced by procarbazine at therapeutic doses. Exposure to bleomycin or procarbazine abolished the characteristic biphasic response to ethidium bromide. The absence of anthra-cycline-induced degradation of Ehrlich ascites tumour cell DNA is apparently in contrast with the DNA damage observed in L1210 tumour cells. These observations suggest that DNA damage is not a necessary condition for antitumour activity.     

Changes in the nucleotide metabolism of Ehrlich ascites tumour cells during their growth in vivo

During the transition of Ehrlich mouse ascites tumour cells from the proliferating into the resting phase of growth a tremendous loss of purine and pyrimidine compounds was quantitated by ion-pair reversed-phase high performance liquid chromatography. This change is accompanied by a distinct decline in the incorporation rates of adenine, hypoxanthine, and adenosine. Inorganic phosphate stimulates the low rate of hypoxanthine incorporation of cells in the plateau phase, but lacks any effect on the high rate during proliferation. The mitochondria suffer structural deteriorations and decrease in their cellular content in the course of the plateau phase; however, other changes were not seen by morphometric analysis. The interrelations between nucleotide metabolism, mitochondrial content and the rates of formation and consumption of ATP are discussed.     

Long-distance interactions between Ehrlich ascites tumour cells.

In previous work, electron micrographs were made of adjacent surfaces of aldehyde-fixed, Ehrlich ascites tumour cells cultured on coverslips, after reacting some of their negatively charged surface sites with colloidal iron hydroxide (CIH) particles. It was observed that microvilli from one cell were aligned with intermicrovillus regions on another, where the density of the adsorbed CIH particles was significantly lower than in adjacent regions. Alignment, which was considered to represent interactions between the two peripheral cellular regions, took place when these regions were apparently separated by more than 200 nm, in an environment of physiologic ionic strength ( 0·145 m NaCl).In this communication we attempt to find feasible mechanisms for the alignment phenomenon in physical terms, in cases where the observed separation of 200 nm is correct, and in cases where the distances are overestimated due to preparative artifacts.It is concluded, that at distances of separation in excess of 200 nm, one feasible mechanism for alignment is that net negatively charged macromolecules diffusing out of cells in the region of their microvilli, electrostatically repel CIH-binding anionic sites in the lipid-rich “fluid” matrix of the periphery of the opposed cell, causing gaps in their distribution. The role of electrostatic and electrodynamic (van der Waals') forces in causing alignment is also discussed in terms of distance of separation.This communication is concerned with the interpretation in terms of various interactions, of electron micrographs showing evidence of alignment between microvilli from one cell with specific areas of another.     

Rejoining of DNA strand breaks by T4 DNA ligase in mammalian cells

We have tested the ability of T4 DNA ligase to rejoin radiation-induced DNA strand breaks in living hamster cells (CHO-K1, EM9, xrs-5). T4 DNA ligase was introduced into cells by electroporation prior to x-irradiation. Single- and double-strand breaks were measured by the alkaline comet assay technique, and double-strand breaks (DSBs) were evaluated by the pulsed-field gel electrophoresis method. In the comet assay, the three cell lines showed reduced tail moments following pretreatment with T4 DNA ligase, both directly after irradiation and after repair incubation for 4 h. Similarly, the results obtained from pulsed-field gel electrophoresis showed reduced DSB frequencies after pretreatment with T4 DNA ligase. We conclude that exogeneous T4 ligase contributes to rejoining of radiation-induced strand breaks.     

Glycolysis and glutaminolysis in perifused Ehrlich ascites tumour cells

A perifusion system was designed in order to study glucose and glutamine metabolism by freshly harvested Ehrlich ascites tumour cells in steady state conditions. Cells were perifused in the presence of 5 mM glucose, 0.5 mM glutamine or 5 mM glucose and 0.5 mM glutamine. The results in steady state reveal that both substrates glucose and glutamine are continuously wasted by tumour cells, excreting two moles of lactate per mol of glucose and one mol of glutamate and ammonia per mol of glutamine consumed into the medium. Glutamine consumption in the presence of glucose was higher than with glutamine alone.     

Cytoplasmic 3H-arginine polypeptides of Ehrlich ascites tumour cells

Summary Low molecular weight ninhydrin positive peptide fractions of the Ehrlich tumour cell cytoplasm were isolated and characterized. After preliminary gel filtration of the cytoplasm on Sephadex G-25 column, the peptide mixture was fractionated on cationic exchanger SP-Sephadex C-25 column and eluted with increasing pH gradient. Five peaks were obtained. Only the first peak contained sugar component. All five peptides were studied with respect to molecular weight, isoelectric point and electrophoretic homogeneity. The cytoplasm of Ehrlich tumour cells contains one peptide of acidic (pI - 5.0), two slightly basic (pI - 7.7 and pI - 7.7) and two strongly basic nature (pI - 8.7 and pI - 8.9). Molecular weights varied from 8 500 to 18 500 daltons. The origin of these peptides is briefly discussed.     

Regulation of anaerobic glycolysis in Ehrlich ascites tumour cells.

1) In intact Ehrlich ascites tumour cells the anaerobic glycolytic flux rate and pattern of intermediates have been investigated at different pH values of the extracellular medium. 2) As predicted from the dependence of the lactic acid dehydrogenase equilibrium on pH a strong negative correlation between log ([lactate]/[pyruvate]) and pH has been found. 3) The steady state fluxes of glycolysis at pH 8.0 and 7.4 are rather equal, despite significant differences in the intracellular concentrations of glycolytic intermediates. At pH 8.0 the concentrations of ATP, glucose 6-phosphate, and fructose 6-phosphate are lower, and the concentrations of ADP, AMP, fructose 1,6-bisphosphate, triose phosphates, phosphoglycerates, and phosphoenolpyruvate are higher than at pH 7.4. 4) From the analysis of the pH dependent changes of metabolites it follows that different mechanisms are responsible for maintaining equal actual activities of hexokinase, phosphofructokinase and pyruvate kinase at pH 7.4 and 8.0. 5) From an application of the linear theory of enzymatic chains and a calculation of the control strength of the regulatory important enzymes results that hexokinase is evidently rate-limiting for glycolysis, and phosphofructokinase is also significantly influencing the glycolytic flux. Pyruvate kinase and glyceraldehyde phosphate dehydrogenase, on the other hand, do not significantly affect the rate of the overall glycolytic flux in ascites.     

Glucose regulates its own transport in Ehrlich ascites tumour cells

The capacity of Ehrlich ascites tumour cells to take up 2-deoxy-D-glucose and to bind cytochalasin B varies adaptatively with the level of glucose in the plasma or culture medium. The effect of glucose is exerted directly on the cells and does not necessarily require the participation of hormones such as insulin, glucagon or corticosterone, although glucagon and the glucocorticoids, but not insulin, can also increase the number of glucose carrier molecules administered in vitro. Cycloheximide suppresses the acute inductive effect of glucose, suggesting that protein synthesis might be required for the increased transport activity.     

Fractionation of the detergent-resistant filamentous network of Ehrlich ascites tumour cells

Previous investigations of the filamentous network in eukaryotic cells have been based on observations by electron and fluorescence microscopy. In order to examined, in more detail, the interconnection of the various components of th filamentous network, we have treated Ehrlich ascites tumour cells with Triton X-100 in the presence of Mg++, disassembled the detergent-resistant, residual cell structure with Tris-EDTA and subjected the postnuclear supernatant to sucrose density gradient equilibrium centrifugation. Using this technique we are able to demonstrate 1) the association of the major part of intermediate-sized filament protein (vimentin) with unfolded ribosomal subunits, 2) the nearly identical sedimentation behavior of the boundary lamina and actin, and a minor part of the intermediate-sized filament protein respectively, and 3) the association of a Ca++-dependent protease specific for vimentin intermediate-sized filament protein with the Triton X-100 resistant, residual cell structure. Furthermore, we are able to confirm, by labelling intact Ehrlich ascites tumour cells with [3H] concanavalin A and recovering radioactivity in the lighter sucrose gradient fractions, that the detergent-resistant boundary lamina is derived from the plasma membrane. The presence of coated vesicles in Triton X-100-treated cells as well as of coated pits in the derived membrane point at the same origin of the boundary lamina. The results of the fractionation study are correlated with structures observed by electron microscopy of ultrathin sections of the intact filamentous network.