Participation of the SSB protein in the repair of the DNA in Ehrlich ascites carcinoma cells following gamma radiation

Survival of Ehrlich ascites tumor cells, SSB content of the chromatin, and repair DNA synthesis rate were investigated after gamma-irradiation, the rate of repair synthesis was shown to depend on the SSB-protein content of the chromatin. Changes in the amount of SSB-protein in the chromatin were not connected with the postirradiation accumulation of single-strand sites in DNA.     

Neutral glycosphingolipids in Ehrlich ascites carcinoma cells

The structure of the neutral glycosphingolipids of the Ehrlich ascite carcinoma (EAC) cells was studied. The main four components were identified as glycosylceramide, lastosylceramide, N-acetylgalactosyllactosylceramide and galactosyl-N-acetyllactosylceramide (asialo-GM1). The neutral glycolipid pattern of the cells was found to depend on their density. Dilution of the cell suspension resulted in an increased content of asia-lo-GM1, whereas the content of the other neutral glycolipids remained unchanged. The possible connection between these changes and the earlier disclosed cell density dependence of the gangliosides in EAC cells is discussed.     

The role of protein binding with single-stranded DNA (SSB-protein) in DNA replication in Ehrlich ascites carcinoma cells

Possible involvement of the single-strand DNA-binding protein (SSB-protein) in DNA replication in Ehrlich ascite tumour (EAT) cells was studied. There was a direct correlation between the content of SSB-protein in chromatin and the intensity of replicative synthesis of DNA in various preparations of EAT in vitro and in vivo (the computed value of the correlation coefficient was equal to 0.9). It was shown that the addition of exogenous SSB-protein to permeable EAT cells increased the replicative synthesis. It was concluded that although eukaryotic SSB-proteins are not complete analogs of prokaryotic ones, they may participate in DNA replication in eukaryotic cells and, possibly, are intracellular regulators of proliferation.     

DNA repair kinetics in mammalian cells following split-dose irradiation

The in situ DNA repair kinetics in intracerebral 9L tumor cells and cerebellar neurons following the second of two 1250- or 2500-rad doses separated by various times have been measured using alkaline sucrose gradients in zonal rotors. For both doses and all times employed, both cell types exhibited biphasic kinetics similar to those observed after single doses. When the two doses were separated by less than 2 hr in neurons (1 hr for tumor cells), the half-time (T1/2) of the slow phase was faster than that expected based on the amount of damage present and remained constant until the observed T1/2 coincided with the expected T1/2. When repair of the damage produced by the first dose was complete, the slow phase after the second dose exhibited the same T1/2 as after a single dose. These results suggest that the accessibility of a fraction of the chromatin is altered for a finite period during the repair process, and upon completion of repair is returned to a state indistinguishable from that existing prior to irradiation.     

Heterogeniety of the amino acid pool for protein synthesis in Ehrlich ascites carcinoma cells

Using a new methodological approach based on a step-wise labelling with [14C] and [3H] amino acids, it was demonstrated that the Ehrlich ascite carcinoma cells are capable of utilizing both intracellular and extracellular amino acid pools for protein synthesis. The inhibition of amino acid transport into the cells is accompanied by a more intensive utilization of the exogenous pool. The described procedure permits to calculate the specific radioactivity of the tRNA-bound amino acid and the absolute rate of protein synthesis.     

The effect of fast electrons and menadione on the structural-functional status of Ehrlich ascites carcinoma cells in the early periods following irradiation

Irradiation of Ehrlich ascites tumor cells stimulates oxygen consumption, and menadione suppresses cell respiration. The combined effect of the two factors produces an additional, in comparison with the effect of menadione alone, inhibition of the rate of oxygen consumption by cells. There is an additive effect of radiation and menadione with regard to the level of cell thiols and interphase cell death.     

The effect of SSB-protein from Ehrlich ascites carcinoma cells on activity of various enzymes of DNA replication and repair

The effect of a single-stranded DNA-binding protein (SSB-protein) form Ehrlich ascites tumour cells (EAT) on the activity of homologous purified DNA-polymerases alpha and beta, DNA-replicase, primase and DNA-polymerases from phage T4 and Bacillus stearothermophillus was studied. It was shown that the SSB-protein caused a 1.5-2.5-fold stimulation of the DNA-polymerase alpha activity on different templates (e.g., denaturated and activated DNA, poly(dA). The degree of stimulation depended on the template type, protein/template ratio and purity of DNA-polymerase alpha. The activity of DNA-polymerase was inhibited by the SSB-protein, when the activated DNA was used as a matrix and was unchanged on the denaturated DNA. The activity of some prokaryotic DNA-polymerases was increased under the influence of the SSB-protein. The protein enhanced the processivity of T4 DNA-polymerase and strongly inhibited the activity of replicase and primase. A conclusion about the complex effect of the SSB-protein on the activity of replicative and repair enzymes is drawn.     

The binding of SSB-proteins with DNA in chromatin of Ehrlich ascites carcinoma cells]

Using UV-induced cross-linking between proteins and DNA, the contacts between single-stranded DNA-binding proteins (SSB proteins) and chromatin DNA have been demonstrated. Ehrlich ascites tumour DNA was labeled in vivo by inoculation of tumour-bearing mice with 3H-thymidine. The cells were irradiated with the UV light dose of 3000 J/m2, destroyed in a Triton X-100-containing hypotonic medium, and separated by centrifugation into the extrachromatin fraction and chromatin. Chromatin DNA was digested with DNAase 1, and the chromatin proteins were extracted with 2 M NaCl-polyethyleneglycol. SSB proteins from the extrachromatin fraction and chromatin were purified. Only SSB proteins from UV-irradiated cell chromatin appeared to possess a high specific radioactivity which exceeded 7.5-fold that of non-irradiated cells. There were no differences between chromatin SSB proteins in control and irradiated cells as could be evidenced from SDS electrophoresis data. It is assumed that in irradiated cells SSB proteins of DNA-digested chromatin are covalently cross-linked with DNA fragments.     

Binding of SSB-protein from Ehrlich ascites carcinoma cells with DNA and polyribonucleotides]

Binding of SSB-protein from Ehrlich ascites tumor to ssDNA from M13 phage leads to its compactization. The structure of the complex at the protein/DNA ratios far from the saturation level looks like "beads-on the string". DNA that was fully saturated with protein forms collapsed globular structure. Binding of the protein to the dsDNA from phage lambda increases its flexibility and decreases the coil dimensions; no "beads-on the string" structure are seen. The protein possess slight destabilizing effect on hairpin helices of M13DNA. Competition studies demonstrate that the binding properties of protein with polyribonucleotide lattices and DNA's decrease in ranking as follows: poly(rG) greater than or equal to poly(rI) greater than or equal to ssDNA greater than dsDNA greater than poly(rA) congruent to approximately poly(rU). Thus SSB-protein from Ehrlich ascites tumor differs significantly from its presumed prokaryotic analogs.     

DNA ligase from mouse Ehrlich ascites tumor cells

The molecular (Mr = 120,000; s20, w = 5S) and catalytic properties (Km (ATP) = 3 microM; Km (nicked DNA) = 0.2 microM; Km (Mg2+) = 3 mM) of DNA ligase from mouse Ehrlich ascites tumor cells are similar to those of the enzymes from calf thymus and rodent liver. The activity level of DNA ligase from the tumor cells is about 10-fold higher than that from mouse liver. Immunochemical titration of DNA ligase with antibodies against the calf thymus enzyme showed that the higher level of DNA ligase activity in the tumor cells is due to an increase in enzyme quantity and not to elevation of the catalytic efficiency of the enzyme molecule. These results suggest that there is little apparent difference between the qualities of DNA ligases from the tumor cells and normal tissues of rodents and calf.     

Oxygen dependence of nuclear DNA replication in Ehrlich ascites cells

Oxygen was excluded from cultured Ehrlich ascites cells for 5-7 h and then readmitted. During the anaerobic period and for about 1 h following reoxygenation the DNA synthesis of the cells was studied by determining the DNA synthesis rate from [3H] thymidine incorporation data, by evaluation of the thymidine (pulse labelling) index, by DNA fibre autoradiography, and by alkaline sucrose gradients in order to follow the maturation of the daughter chains. The DNA synthesis rate was found to decay to a few percent of the initial value within 5-7 h after deoxygenation. Immediately after reoxygenation it increased to exceed the control level within 0.5-1 h. The only partial process of the genome replication definitely responding to deoxygenation/reoxygenation was the initiation of new replicon units, while progress of the replication forks and maturation of the new daughter chains were not significantly affected. The coordination of replicon initiation within groups or clusters was maintained throughout. The interruption of replication at the level of initiation of clusters upon deoxygenation was interpreted as a regulatory response of the cells to ensure basic viability under unfavourable conditions.     

DNA polymerases in isolated 'nuclear matrix' of Ehrlich ascites cells

Isolated nuclei from Ehrlich ascites tumor cells continue a replicative-like in vitro DNA synthesis. Polymerase alpha is the major dNTP polymerizing enzyme in nuclei. Following complete achromatinization dNTP polymerizing activities are still associated with the residual structure termed 'nuclear matrix'. In contrast to DNA synthesis in native nuclei, 'nuclear matrix' DNA synthesis is mainly due to polymerase beta-like activity.