Repair of potentially lethal radiation damage in confluent and non-confluent cultures of human hybrid cells

The repair of potentially lethal damage (PLDR) in a gamma-irradiated human hybrid cell line (skin fibroblast X HeLa) and its tumourigenic segregant has been studied as a function of cell density at the time of irradiation and during the postirradiation repair period. The data show that PLDR occurs in both non-confluent and confluent cultures of both cell lines. Furthermore, there is evidence that the extent of PLDR is dependent on cell density and that cell-cell contact may be an important factor in this regard.     

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.     

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.     

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.     

Electrophoretic mobility of irradiated erythrocytes and Ehrlich ascites tumour cells.

The subunits of RNA polymerase I are partially resolved during density gradient centrifugation. An analysis of the relative subunit composition with respect to specific catalytic activity shows that the molar ratio of the 24,000 dalton subunit directly correlates with polymerase activity. Since this polypeptide is found also in polymerases II and III, it may be required for activity of all yeast nuclear RNA polymerases.     

Repair of potentially lethal damage in yeast induced by fast neutrons

Survival curves of 3 diploid (D7) yeast strains: one wild-type, one deficient in excision of pyrimidine dimers (UV-sensitive) and one blocked in DNA double-strand-break repair (X-ray-sensitive), were compared after irradiation with cyclotron-produced fast neutrons. It was observed that both the UV-sensitive (rad3/rad3) and the X-ray-sensitive (rad52/rad52) mutants were more sensitive to neutrons than the wild-type. The role of DNA double-strand-breaks in neutron-induced cell death was further studied by comparing the relative sensitivity of the rad52/rad52 mutant to gamma-rays and fast neutrons. A comparison of the dose modification factors revealed that the deficiency in DNA double-strand-break repair did not make the yeast cells more sensitive to neutrons than to photons, which suggests that lesions of a different type may also be produced by neutrons. Survival curves obtained upon immediate plating and after delayed plating of neutron-irradiated cells showed that all 3 yeast strains were efficient in liquid holding recovery. The role of different repair pathways in cellular recovery from neutron-induced lethal damage is discussed.     

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.     

Zinc-, copper- and cadmium-binding protein in Ehrlich ascites tumour cells.

The properties of Ehrlich ascites tumour cells exposed in vivo to cadmium were investigated as a function of the zinc status of the host animals. Tumour-cell growth was inhibited by cadmium in both zinc-sufficient and zinc-deficient animals. However, cells in zinc-sufficient tumours accumulate much less cadmium than those in deficient tumours. The subcellular distributions of cadmium and zinc do not depend on zinc status. Cadmium and zinc are bound to a low-molecular-weight protein with properties similar to metallothionein. Without exposure to cadmium, a zinc- and copper-binding protein is still present that behaves like a metallothionein. This protein can rapidly bind cadmium added to Ehrlich cells in vitro. It is shown that the zinc- and copper-binding protein contains free thiol groups. Ehrlich cells isolated from cadmium-treated animals are viable and show normal incorporation of uridine into RNA, but the cellular uptake of thymidine and its incorporation into DNA are inhibited.     

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.     

Repair of potentially lethal damage does not depend on functional TP53 in human glioblastoma cells

The functionality of G(1)-phase arrest was investigated in relation to repair of potentially lethal damage (PLD) in human glioblastoma Gli-06 cells. Confluent cultures were irradiated and plated for clonogenic survival either immediately or 24 h after gamma irradiation. Bivariate flow cytometry was performed to assess the distribution over the cell cycle. Levels of TP53 and CDKN1A protein were assessed with Western blotting and levels of CDKN1A mRNA with RT-PCR. Confluence significantly reduced the number of proliferating cells. Marked PLD repair was found in the absence of an intact G(1) arrest. No accumulation of TP53 was observed, and the protein was smaller than the wild-type TP53 of RKO cells. No increased expression of CDKN1A at the mRNA or protein levels was found in Gli-06 cells. The TP53 of Gli-06 cells was unable to transactivate the CDKN1A gene. From this study, it is evident that PLD repair may be present without a functional TP53 or G(1) arrest.