PubMed Health⌕ Search

Biomedical subjects

E Kaminskas

Publications and source records attributed to E Kaminskas.

At least 19 recordsLinked to original sources
PubMed1992

Repair of DNA damage induced by oxygen radicals in human non-proliferating and proliferating lymphocytes.

Repair of DNA lesions induced by oxygen radicals, generated by xanthine/xanthine oxidase (X/XO), was studied in human peripheral blood lymphocytes and in PHA-stimulated proliferating lymphocytes from 4 healthy subjects. The lesions included DNA-strand breaks (SSB) and other lesions that are converted to SSB under alkaline conditions. The frequencies of SSB were estimated by fluorometric analysis of DNA unwinding. Maximum production of SSB occurred within 10 min of incubation with X/XO at 22 degrees C; with 0.5 mM or higher concentrations of xanthine; and with 0.1-0.5 units/ml of xanthine oxidase. Proliferating lymphocytes repaired X/XO-induced SSB about 4 times more rapidly than lymphocytes. Lymphocytes repaired X/XO-induced SSB more slowly than SSB caused by gamma-radiation. These findings are consistent with the evidence that a number of DNA-repair enzymes have greater activity in proliferating cells than in resting cells. These findings also support the view that there are differences between the DNA damage due to oxygen radicals and that due to ionizing radiation.

DNA↗
PubMed1989

DNA fragmentation in permeabilized cells and nuclei. The role of (Ca2+ + Mg2+)-dependent endodeoxyribonuclease.

Permeabilized mammalian cells and isolated nuclei were used to study various aspects of DNA replication and repair. The present paper describes a progressive fragmentation of parental DNA in human lymphoblastoid cells that were permeabilized with L-alpha-lysophosphatidylcholine or with saponin and incubated at 37 degrees C in a DNA-synthesis mixture. The formation of DNA single-strand breaks (measured by alkaline elution) was linear with the time of incubation and was temperature-dependent. It was prevented by deleting Mg2+ or both Mg2+ and Ca2+ from the incubation mixture, or by the addition of EDTA. It was increased by deleting the components necessary for DNA synthesis, and by substituting Mn2+ for Mg2+ and Ca2+. DNA strand breaks also accumulated in isolated nuclei incubated in a DNA synthesis mixture, but not when Mg2+ was omitted. These results suggest that DNA fragmentation in permeabilized cells and nuclei was due to an activation of (Ca2+ + Mg2+)-dependent endodeoxyribonucleases. The integrity of template DNA needs to be ascertained when the conditions for measuring DNA synthesis in permeabilized cells or in nuclei are formulated.

Cell Line↗
PubMed1987

Progressive formation of DNA lesions in cultured Ehrlich ascites tumor cells treated with hydroxyurea.

We have previously demonstrated an accumulation of strand breaks in mature DNA of cultured Ehrlich ascites tumor cells treated with methotrexate. We postulated that the strand breaks arose from unrepaired spontaneous DNA lesions. The present study describes a progressive accumulation of strand breaks in mature DNA of Ehrlich ascites cells treated with hydroxyurea (HU). Strand breaks were determined by alkaline elution. Accumulation of strand breaks was dependent on the length of incubation (0-16 h) and on HU concentration (0-10 mM). About 70% of strand breaks were repaired when cells were incubated without HU. About 67% of strand breaks were prevented by 0.4 mM deoxyadenosine, deoxyguanosine, and deoxycytidine, with or without thymidine. Prevention was less effective by deoxyadenosine and deoxyguanosine and ineffective by deoxycytidine. Free radical scavengers did not prevent strand breaks. S-phase cells accumulated about twice the number of strand breaks as non-S-phase cells. Cell survival decreased in proportion to the increase in HU concentration (0-10 mM). The results demonstrate that lack of purine, as well as of pyrimidine, nucleotides results in strand breaks in mature DNA, suggest that HU cytotoxicity is due to fragmentation of mature DNA, and caution against the use of HU in DNA repair studies.

Animals↗
PubMed1985

Deficient repair of DNA lesions in Alzheimer's disease fibroblasts.

DNA strand breaks, resulting from treatment with N-methyl-N'-nitro-N-nitrosoguanidine, were repaired more slowly in four strains of familial Alzheimer's disease fibroblasts than in five strains of fibroblasts from age-matched normals. These results were not due to differences between the two cell types in in vitro ages, in the initial DNA damage or in drug-induced cell lysis. Bleomycin-induced DNA double-strand breaks were repaired equally efficiently by both types of cells. Alzheimer's disease cells may have a DNA repair defect, which may be involved in the pathogenesis of this disease.

Aged↗
PubMed1984

Accumulation of DNA strand breaks and methotrexate cytotoxicity.

There was a progressive formation of strand breaks in mature DNA of Ehrlich ascites tumor cells that were treated with methotrexate. Cells were labeled with [14C]thymidine before incubation with methotrexate, and DNA strand breaks were measured by alkaline and by neutral filter elution methods. DNA single-strand breaks accumulated in a linear fashion as a function of time during the first 10 hr of incubation with 2 microM methotrexate. Thereafter, the accumulation of DNA strand breaks deviated from linearity because of progressive cell death. The extent of DNA strand breakage in cells that had been incubated with methotrexate for 24 hr was as high as in cells that had been irradiated with 300 rads. DNA strand breaks persisted in cells that were incubated, after exposure to methotrexate, in medium containing thymidine, hypoxanthine, and nonessential amino acids, indicating that these strand breaks were poorly repaired. Cell death commenced after 10 hr of incubation with methotrexate and continued during the following 3-4 days. These findings suggest that cell death was due to a lethal accumulation of DNA strand breaks. The formation of DNA strand breaks is probably due to inefficient DNA repair, resulting from the inhibition of syntheses of thymidylate and of purine nucleotides. The accumulation of DNA strand breaks was minimal in growth-arrested cells, which are resistant to methotrexate toxicity.

Animals↗
PubMed1982

Effects of methotrexate on ribonucleotide pools in growing and in growth-arrested tumor cells and antagonism by RNA synthesis inhibitors.

We examined the mechanism of ribonucleotide pool changes in methotrexate-treated Ehrlich ascites tumor cells in culture. In exponentially growing cells, methotrexate inhibited ATP and GTP formation and stimulated UTP and CTP formation by the de novo pathways. Over the following 4 h, ATP and GTP decreased and UTP and CTP increased. During this period RNA synthesis rates decreased by 30 to 40%. Inhibitors of nucleolar RNA synthesis (actinomycin D, doxorubicin, or cycloheximide), but not of DNA synthesis (cytosine arabinoside), prevented ATP and GTP depletion. These results suggest that ATP and GTP were depleted in methotrexate-treated cells because the shift-down of RNA synthesis occurred more slowly than the inhibition of ATP and GTP supply. UTP and CTP pools increased because of increased de novo synthesis and because of decreased consumption in RNA synthesis. Ribonucleoside triphosphates were increased and their syntheses by de novo pathways were inhibited in cells treated with RNA synthesis inhibitors. ATP decreased slightly and GTP remained unchanged in growth-arrested cells treated with methotrexate. In growth-arrested cells, de novo purine nucleotide synthesis was inhibited less and RNA synthesis rates were lower than in growing cells.

Animals↗
PubMed1978

The pH-dependence of sugar-transport and glycolysis in cultured Ehrlich ascites-tumour cells.

1. pH-dependence of glycolysis has generally been ascribed to the effects of pH on the activities of glycolytic enzymes. The present study shows that sugar transport is pH-dependent in cultured Ehrlich ascites-tumour cells. 2. The rates of glucose consumption, of 3-O-methylglucose transport, and of 2-deoxyglucose transport and phosphorylation increased as linear functions of pH, as the pH of the cell culture medium was increased from 6.1 to 8.5. Transport of glucose, as measured in ATP-depleted cells, was pH-dependent to the same extent as transport of the non-metabolizable sugars. 3. Glucose consumption rates were about 8-fold higher at pH 8.5 than at pH 6.4. About 65-85% of glucose was converted into lactate. Sugar transport rates were 2.5-fold higher at pH 8.5 than at pH 6.3. 4. pH affected both simple diffusion and facilitated diffusion. pH effect was mainly on the Vmax. of 2-deoxyglucose uptake, and on the rapid-uptake phase of 3-O-methylglucose transport. 5. It was estimated that about 70% of the pH effect on the rates of glucose consumption may be due to the effect on sugar transport and the remainder to the effect on the activities of glycolytic enzymes.

Biological Transport↗
PubMed1976

Cyclic AMP and growth of Ehrlich ascites tumor cells. Lack of cyclic AMP elevation in nutritionally deprived cells and mechanism of retardation of growth by dibutryl cyclic AMP.

Cyclic AMP levels in Ehrlich ascites tumor cells changed little after deprivation of cells of essential nutrients, serum, glucose and amino acids, deprival of each of which leads to marked inhibition of growth and protein synthesis. Cyclic AMP levels also changed little after the addition of these nutrients to deprived cells. Thus cyclic AMP is not likely to be the intracellular mediator for growth regulation by these three nutrients. Elevation of cyclic AMP levels for short periods by exposure of cells to choleratoxin or theophylline produced only slight changes in parameters of protein synthesis (polyribosome pattern and rate of [3H]leucine incorporation). An exposure for 1 day to dibutyryl cyclic AMP did not inhibit cell growth. However, prolonged exposure to dibutyryl cyclic AMP inhibited the multiplication of Ehrlich ascites cells both in suspension and in stationary cultures. No morphological effects were evident in the former; in the latter, cells attached firmly to the substratum and formed elongated cytoplasmic processes. Inhibition of cell multiplication by dibutyryl cyclic AMP was related to cell density and to serum concentration. Cells in dibutyryl cyclic AMP-containing media plated at low cell densities multiplied as rapidly as control cells. The final densities cells reached were determined by the serum concentration; in dibutyryl cyclic AMP-containing media these densities were about one-half those of respective control cells. Limitation of cell multiplication by dibutyryl cyclic AMP was reversed by the addition of serum, by resuspending cells at lower densities, or by resuspending cells in media without dibutyryl cyclic AMP. These findings suggested that dibutyryl cyclic AMP may affect the utilization of serum factors by cells. Dibutyryl cyclic AMP did not inactivate serum factors and did not change the rate at which cells depleted the growth medium of serum factors. Dibutyryl cyclic AMP may limit cell multiplication by increasing the cellular requirement for serum factors.

Amino Acids↗
PubMed1975

Changes in adenylate energy charge in Ehrlich ascites tumor cells deprived of serum, glucose, or amino acids.

The adenylate energy charge in Ehrlich ascites tumor cells increases when cells are cultivated in serum-limiting medium and decreases when they are incubated in glucose- or amino acid-limited media. Protein synthetic rates decrease in cells deprived of serum, glucose, or amino acids. Supplementation of deprived cells with respective nutrients restores normal protein synthetic rates and adenylate energy charge values. Serum-deprived cells incubated in depleted serum media do not increase their rates of protein synthesis and their adenylate energy charge remains elevated. These results suggest that serum factors regulate protein synthetic rates by mechanisms other than those regulating the availability in cells of glucose or of amino acids. The increased rates of utilization of glucose and of amino acids following the addition of serum are probably due to increased biosynthetic requirements.

Adenine Nucleotides↗