DNA polymerase in nucleoli isolated from Ehrlich ascites tumor cells

DNA replication was investigated in nucleoli isolated from Ehrlich ascites tumor cells. DNA synthesis was dependent on the presence of the four deoxynucleoside triphosphates and magnesium, but was reduced in the presence of ATP. The pH optimum for DNA replication was 8.5 to 9.0 N-Ethyl-maleimide reduced the reaction significantly. DNA synthesis occurred on nucleolar chromatin and was stimulated by treatment of the nucleoli with a small amount of DNase I. Addition of exogenous DNA to the reaction mixture significantly stimulated [³H]dTMP incorporation.     

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.     

Primer specificity of ribosome-associated poly(A) polymerase from Ehrlich ascites tumour cells

The reaction product of the ribosomal poly(A) polymerase [ATP(UTP):RNA nucleotidyltransferase] is analyzed. Two systems are used in vitro: (a) isolated polyribosomes with endogenous enzyme and RNA primer and (b) purified enzyme with total polyribosomal RNA as primer. In the polyribosome system about 50% of the [3H]AMP label is in poly(A)-containing mRNA. This RNA displays a heterogeneous size ditribution in the range of 8--30 S with a maximum at about 14 S. Upon denaturation the maximum is shifted towards the 10-S zone. The poly(A) polymerase catalyzes the addition of 12--18 adenylate residues to pre-existing mRNA poly(A) sequences of 40--160 residues. The [3H]AMP incorporated into poly(A)-lacking RNA is mainly in a fraction with an electrophoretic mobility corresponding to 4-S RNA. In the purified enzyme system, specificity towards poly(A)-containing mRNA is lost to a considerable extent. Only 10% of the [3H]AMP label is retained by oligo(dT)-cellulose. The bulk of the product is in 18-S rRNA and heterogeneous small molecular weight RNA. We conclude that the ribosome-associated poly(A) polymerase is most likely the enzyme responsible for the cytoplasmic polyadenylation of poly(A)-containing mRNA in vivo.     

Mitoxantrone toxicity on Ehrlich ascites tumour cells: inhibition of the transplasma membrane redox activity

This study focused on the potent cytotoxic effect that mitoxantrone produces on Ehrlich ascites tumour cells. Host mice treated with mitoxantrone showed a life span three times higher than control non-treated host mice. Mitoxantrone also showed a potent cytotoxic effect on Ehrlich cells incubated in vitro for only a few hours. Studies on the effect of mitoxantrone on a plasma membrane redox system showed that mitoxantrone inhibits this activity, which is apparently related to cell proliferation.     

Studies on DNA content, RNA synthesis, and DNA template activity in aging cells of Paramecium aurelia.

Investigations by Feulgen microspectrophotometry in Paramecium aurelia indicated that as fission age increased the amount of macronuclear DNA decreased. It was also found that the amount of RNA synthesis as determined by the in vivo incorporation of [³H]uridine decreased as the fission age increased. An alternative in situ assay of the DNA template activity determined by the RNA polymerase-catalyzed incorporation of [³H]UTP is described. The DNA template activity of older cells was shown to be significantly lower on a per cell basis than that of younger cells. The majority of this reduction was shown to be due to the gradual loss of DNA template with an increase in fission age. The specific activity of the DNA template, however, does show a small but significant decrease as the fission age of the cell increases.     

Stimulation of protein synthesis by lysine analogues in lysine-deprived Ehrlich ascites tumour cells

This paper describes experiments in which we have investigated the mechanism by which amino acid starvation regulates the initiation of protein synthesis in mammalian cells. We have examined the ability of a range of lysine analogues to stimulate protein synthesis in lysine-deprived mouse Ehrlich ascites tumour cells in culture. Of those analogues tested, only those which are cleaved to lysine intracellularly are capable of restoring protein synthesis to the level seen in fully fed cells. Lysine which is covalently linked to agarose does not stimulate translation. After 5 min incubation of lysine-deprived cells with the analogue lysine p-nitroanilide, the lysine concentration in cell extracts is restored to that found in extracts from fed cells, and protein synthesis is maximally stimulated within 5–10 min. During this period of time there is no increase in the concentration of lysine in the medium. These data indicate that it is the size of the intracellular rather than the extracellular amino acid pool which regulates the rate of protein synthesis during amino acid deprivation.     

Lack of correlation between thymidine kinase activity and changes of DNA synthesis with tumour age: an in vivo study in Ehrlich ascites tumour

Thymidine kinase (TK) and its isoenzymes were studied in relation to age of Ehrlich ascites tumour cells growing in vivo. Various steps of the pathway of thymidine through deoxynucleotide metabolism were studied: [3H]-thymidine cellular uptake and incorporation into DNA; the cellular nucleotide pools; and the concentration of thymidine in ascites. In addition, the proportion of cells in the various parts of the cell cycle and the bromodeoxyuridine labelling index were determined. Four isoenzymes at pI 4.1, 5.3, 6.9 and 8.3 were identified using isoelectric focusing. The TK activity declined with age of the tumour by about 90%, mostly due to a decrease of the isoenzyme at pI 8.3. However, this decline was neither related to the changes in DNA synthesis rate of the cells with tumour age, nor to the proportion of cells in S-phase or the bromodeoxyuridine (BrdU) labelling index. In contrast, the contribution of DNA synthesis via the thymidine salvage pathway relative to the total DNA synthesis increased from less than 1% at exponential growth to about 15% at plateau phase of growth. Blocking of DNA synthesis by aphidicolin did not change the TK activity. We therefore conclude that changes in TK activity and changes in cell growth are epiphenomena rather than causally related to each other. All nucleotide pools decreased with tumour age. The inhibition of TK by an increase in the deoxythymidine triphosphate pool could therefore be excluded. With a decrease of the TK activity during tumour growth, increasing amounts of TdR were excreted by the cells and accumulated in the ascites fluid. To explain our results on TK activity we propose a substrate cycle in which thymidine monophosphate supplied by de novo synthesis is dephosphorylated and is then either phosphorylated by TK to thymidine monophosphate or excreted by the cell.     

Lack of correlation between thymidine kinase activity and changes of DNA synthesis with tumour age: an in vivo study in Ehrlich ascites tumour

Abstract. Thymidine kinase (TK) and its isoenzymes were studied in relation to age of Ehrlich ascites tumour cells growing in vivo. Various steps of the pathway of thymidine through deoxynucleotide metabolism were studied: [³H]-thymidine cellular uptake and incorporation into DNA; the cellular nucleotide pools; and the concentration of thymidine in ascites. In addition, the proportion of cells in the various parts of the cell cycle and the bromodeoxyuridine labelling index were determined.
Four isoenzymes at pi 41, 5-3, 6–9 and 8-3 were identified using isoelectric focusing. The TK activity declined with age of the tumour by about 90%, mostly due to a decrease of the isoenzyme at pi 8-3. However, this decline was neither related to the changes in DNA synthesis rate of the cells with tumour age, nor to the proportion of cells in S-phase or the bromodeoxyuridine (BrdU) labelling index. In contrast, the contribution of DNA synthesis via the thymidine salvage pathway relative to the total DNA synthesis increased from less than 1% at exponential growth to about 15% at plateau phase of growth. Blocking of DNA synthesis by aphidicolin did not change the TK activity. We therefore conclude that changes in TK activity and changes in cell growth are epiphenomena rather than causally related to each other.
All nucleotide pools decreased with tumour age. The inhibition of TK by an increase in the deoxythymidine triphosphate pool could therefore be excluded. With a decrease of the TK activity during tumour growth, increasing amounts of TdR were excreted by the cells and accumulated in the ascites fluid. To explain our results on TK activity we propose a substrate cycle in which thymidine monophosphate supplied by de novo synthesis is dephosphorylated and is then either phosphorylated by TK to thymidine monophosphate or excreted by the cell.     

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.     

Glutamine and glucose as energy substrates for Ehrlich ascites tumour cells

Energy metabolism of freshly harvested Ehrlich ascites tumour cells in the presence of 5 mM glucose and/or 0.5 mM glutamine was studied. The rate of oxygen utilization was not altered by the addition of 0.5 mM glutamine; 5 mM glucose induced an inhibition of respiration. In the presence of both glucose and glutamine, the Crabtree effect decreased. In these conditions, the rates of oxygen uptake, the CO2 evolution and the changes in the redox states of cytochromes indicate that glucose is preferred by Ehrlich ascites tumour cells as energy substrate. Glucose decreased the rate of glutamine utilization by 34%. On the other hand, glutaminolysis did not inhibit glycolysis.     

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.     

In vitro conversion of MVM parvovirus single-stranded DNA to the replicative form by DNA polymerase alpha from Ehrlich ascites tumour cells.

A partially purified preparation of DNA polymerase alpha, obtained from the cytosol of Ehrlich ascites tumour cells, has been found to catalyze the conversion of MVM parvovirus, SS DNA (5 kilobases) to RF in vitro. The reaction initiates at a natural 55 base pair hairpin which exists at the 3' terminus of MVM SS DNA. The SS leads to RF conversion is sensitive to aphidicolin, resistant to ddTTP and is promoted by purine ribonucleoside 5' triphosphates, a phenomenon which could not be explained simply by stabilization effects on the in vitro deoxynucleotide precursor pool. In the absence of rNTPs, nascent complementary strands frequently terminate prematurely at a preferred location, between 1300 and 1700 nucleotides from the initiating 3' hairpin terminus. This in vitro system, involving self-primed parvovirus DNA synthesis, provides a convenient assay for those components of the mammalian replicative DNA polymerase complex which are required for the elongation of nascent DNA chains.