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K Overgaard-Hansen

Publications and source records attributed to K Overgaard-Hansen.

13 recordsLinked to original sources
PubMed1997

Expansion of the cellular content of ribonucleoside triphosphates induces cell shrinkage and KCl loss in Ehrlich ascites tumor cells and in Chinese hamster ovary cells.

The conversion to corresponding triphosphate derivatives of various ribonucleosides has been studied in Ehrlich ascites tumor cells and in Chinese hamster ovary cells under conditions that are optimal for cellular uptake of orthophosphate. The initial cellular uptake of orthophosphate is followed by a cellular loss of Cl- which might be consistent with a H2PO4-/Cl- exchange mechanism. Subsequent addition of ribonucleosides to the medium leads to cellular accumulation of the corresponding triphosphate and to a concomitant loss of KCl and to sustained cell volume reduction. The latter two events are quite unspecific with regard to the nucleobase moiety of the ribonucleoside triphosphate accumulated (adenine, guanine and purine being almost equally effective) and they depend in a rather simple way on the increase of the cellular content of these compounds. The KCl loss seems to depend on opening of the separate K+ and Cl- channels. The pharmacological profile of the putative ion channels could not be identified in spite of experiments with conventional blockers. In the case of purine riboside the accumulation of the corresponding triphosphate and concomitant loss of KCl and cell water may be followed by a regain of cell volume due to a continued purine riboside triphosphate accumulation, which apparently depends on the uptake of orthophosphate by cotransport with Na+ and which for osmotic reasons is accompanied by the uptake of water and hence volume increase. The possibility that the nucleoside triphosphate induced opening of a putative Cl- channel may be due to a direct effect of triphosphate on a channel protein is discussed.

Adenine↗
PubMed1996

The effect of 3'-deoxyadenosine N(1)-oxide on growth in vitro and in vivo on Ehrlich ascites tumor and on a human squamous lung cell carcinoma xenograft in nude mice.

The effect of 3'-deoxyadenosine N(1)-oxide (3'-dANO) on Ehrlich ascites tumor and a human squamous lung cell carcinoma was investigated. The 3'-dANO concentration that inhibited the cell growth 50% (IC(50)) in Ehrlich ascites tumor cells in vitro was 0.15 mM, and the killing efficiency concentration (concentration of the drug that kills all cells) was 1 mM. By simultaneous administration of 3'-dANO and the adenosine deaminase inhibitor erythro-9-(2-hydroxyl-3-nonyl) adenine (EHNA), the IC(50) of 3'-dANO was unchanged, but the killing efficiency concentration of 3'dANO was reduced to 0.3 mM. When mice bearing Ehrlich ascites tumor were treated i.p. with 3'-dANO doses of 200 mg/kg daily for 4 days, the mean increased life span (ILS) was 200%. 3'-dANO in combination with EHNA did not further increase the life span of the tumor-bearing mice. The specific growth delay (SGD) of the Ehrlich tumor and of a human squamous lung cell carcinoma growing subcutaneously in 3'-dANO-treated mice were calculated from Gomperts tumor growth curves. The Ehrlich tumor-bearing mice received 3'-dANO i.p. at doses of 250 mg/kg daily for 4 days, and the nude mice bearing human carcinoma received 3'-dANO i.p. at doses of 225 mg/kg daily for 5 days. The SGD for the investigated tumors were calculated to be 1.0 and 1.1, respectively.

Animals↗
PubMed1994

Net uptake of orthophosphate in Ehrlich ascites tumor cells in the presence of purine riboside may be rate limiting for the expansion of the pool of ribonucleotides.

Incubation with adenosine or with structural analogs thereof may in several cell types under some conditions result in the cellular accumulation of abundant amounts of the corresponding triphosphates. In the present work we have found that incubation of cells at high concentrations of orthophosphate (Pi) results in increased intracellular levels thereof, although they become not as high as the extracellular concentration. In the presence of purine riboside (nebularine, Pr) and high concentration of Pi the intracellular Pi is, however, kept at a low steady-state level, probably because it immediately upon uptake is being trapped primarily as the triphosphate of purine riboside. The latter compound accumulates at a constant rate for at least 1 h. The rate of accumulation of the sum of phosphate residues present in Pi, adenosine phosphates and purine riboside phosphates appears to be proportional to the extracellular concentration of Pi and to be highly dependent on pH (6.5 and 7.0 being optimal and 7.9 nonpermissible) but it is unaffected by substitution of Na+ by choline.

Animals↗
PubMed1993

Different relationships between cellular adenosine or 3'-deoxyadenosine phosphorylation and cellular adenine ribonucleotide catabolism may be obtained.

Treatment of BALB/c-3T3 mouse fibroblasts with 3'-deoxyadenosine led to a rapid accumulation of 3'-deoxyadenosine phosphates and the kinetics of this process has been determined. Concomitant with accumulation of these compounds, the adenine ribonucleotide pool was reduced. The kinetics of the two processes suggested that they were tightly coupled. The inhibitory effect of relatively high concentrations of coformycin indicated that IMP was an intermediate in the catabolic pathway. Similar experiments with Ehrlich ascites tumor cells were performed in Ringer-Hepes solution at pH 6.5 or 7.5 and with varying concentrations of orthophosphate. The experiments were performed with cells where ATP was [3H]-labeled. This allowed the determination of the catabolism of adenine ribonucleotides to labeled nucleosides under conditions where added adenosine was phosphorylated. The results showed that at low phosphate concentration (5.8 mM) at pH 6.5 adenosine may be phosphorylated at a rate that was completely balanced to the concomitant catabolism of adenine ribonucleotides; that is, there was apparently a tight kinetic coupling between anabolism of adenosine and catabolism of adenine ribonucleotides. With 3'-deoxyadenosine a corresponding effect was obtained although the apparent coupling between phosphorylation of 3'-deoxyadenosine and catabolism of adenine ribonucleotides was not complete. When experiments were performed at the same pH but at high concentration of phosphate (45 mM) there was in contrast no coupling between the two processes; that is, ATP was present in constant amounts while 3'-deoxyadenosine phosphates accumulated at a high rate. In experiments with adenosine under these conditions there was still some although a relatively limited degree of apparent coupling between phosphorylation of adenosine and catabolism of adenine ribonucleotides. In both lines of cells used and with both adenosine and 3'-deoxyadenosine, the main products of the catabolism of adenine ribonucleotides were inosine and hypoxanthine. With 3'-deoxyadenosine there was in addition (about 20%) formation of xanthosine, suggesting that IMP dehydrogenase had also been activated. These results lead to the suggestion that adenosine (or 3'-deoxyadenosine) may be phosphorylated in two ways. 1) Phosphorylation may depend on an adenosine kinase unrelated to catabolism of adenine ribonucleotides. 2) Phosphorylation may be tightly coupled to catabolism of adenine ribonucleotides. A nucleoside phosphotransferase may catalyze the transfer of a phosphoryl group from IMP to adenosine (or 3'-deoxyadenosine) to form AMP (or 3'-dAMP) and inosine, a process that may be tightly coupled to an AMP deaminase reaction. The IMP formed in the latter reaction may not be released but transferred to the phosphotransferase.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗
PubMed1992

Toxicity and metabolism of 3'-deoxyadenosine N1-oxide in mice and Ehrlich ascites tumor cells.

The toxic effect of 3'-deoxyadenosine N1-oxide (3'-dANO) on mice, on their different organs, and on Ehrlich ascites tumor cells was studied. In both healthy and tumour-bearing animals, the lethal dose for 10% of the mice receiving i.p. injections (LD10) of 3'-dANO was estimated to be about 300 mg/kg x 4 days in one mouse strain (Theiller). In another mouse strain (NMRI), we obtained a markedly higher LD10 value (675 mg/kg x 5 days). At nonlethal doses (250 mg/kg x 4 days), we observed reversible neurological symptoms on days 4-12 after treatment, but no macroscopical or microscopical changes was detected in the brain, heart, thymus, lung, lymph node, spleen, liver, kidney, bone marrow, or gastrointestinal tract. At doses of 450 mg/kg x 4 days, severe neurological symptoms were observed, and atony of the gastrointestinal canal and damage to the kidney and liver were registered. Even at doses that were lethal to the mice, no histopathological change was observed in the bone marrow or in the gastrointestinal canal. Pharmacokinetics studies showed that after the i.p. injection of 3'-dANO, the maximal plasma concentration was reached after 10 min, after which it declined showing a half-life of about 40 min. A transient accumulation of 3'-deoxyadenosine triphosphate (3'-dATP) was observed within 24 h in the liver and kidney, with the maximal concentration being reached after about 2-3 h. 3'-dANO was excreted partly as the unchanged substance and partly as the metabolite 3'-deoxyinosine within 24 h. Flow-cytometric DNA analysis of Ehrlich tumor cells treated either in vitro or in vivo with 3'-dANO revealed no therapy-induced change in the cell-cycle perturbations, which indicates that cells were randomly killed during all phases of the cycle.

Animals↗
PubMed1988

Synergistic effect of 3'-deoxyadenosine N1-oxide and adenosine deaminase inhibitors on growth of Ehrlich ascites tumor cells in vivo.

The simultaneous administration of 3'-deoxyadenosine N1-oxide (3'-dANO) and the adenosine deaminase inhibitors erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) or 2'-deoxycoformycin (2'-dCF) to mice bearing Ehrlich ascites tumor cells resistant to 3'-dANO resulted in 80%-90% inhibition of tumor growth in vivo. 3'-dANO and 2'-dCF increased the survival time of tumor-bearing mice by a factor of 2. In vitro studies showed that the 3'-dANO resistant Ehrlich cells initiate the metabolism of 3'-dANO by a reduction to 3'-deoxyadenosine, which is converted primarily to 3'-deoxyinosine by adenosine deaminase and, to a small extent, phosphorylated to the cell toxic agent 3'-dATP. By the addition of EHNA or 2'-dCF it was possible to block the formation of 3'-deoxyinosine, resulting in a profound stimulation in the accumulation of 3'-dATP. The development of resistance to 3'-dANO was studied in cell cultures and found to be accompanied by changes in the enzyme activities of the reductase, the adenosine kinase, and the adenosine deaminase.

Adenine↗
PubMed1987

Studies on the mechanism of cytotoxicity of 3'-deoxyadenosine N1-oxide in different strains of Ehrlich ascites tumor cells.

The correlation between the metabolic processing of 3'-deoxyadenosine N1-oxide (3'-dANO) in vitro and its effect on tumor growth in vivo has been investigated in seven different strains of Ehrlich ascites tumor cells. The metabolism of 3'-dANO is initiated by reduction to 3'-deoxyadenosine (3'-dA). This process is the rate-limiting process. The 3'-dA does not accumulate, but is converted to 3'-deoxyadenosine triphosphate (3'-dATP) or 3'-deoxyinosine (3'-dI). The ratio between 3'-dATP and 3'-dI inosine corresponds to the ratio between the activities of adenosine kinase and adenosine deaminase in the cell. Two of the cell lines were markedly inhibited by 3'-dANO in vivo. In these cells the accumulation of 3'-dATP was 1.4-2.2 nmol/h per mg cells, which accounts for the major part of the metabolized 3'-dANO. Five of the cell lines were not inhibited by 3'-dANO and the formation of 3'-dATP was 5-10 times less in these than in the sensitive strains. The low level of 3'-dATP is caused primarily by a low ratio between the activities of adenosine kinase and adenosine deaminase, which is 15 time less than in the sensitive cell lines. The rate of reduction of 3'-dANO seems to be of minor importance. These results indicate a correlation between the inhibition of tumor growth by 3'-dANO and the ability of the cell to accumulate 3'-dATP from 3'-dANO and show that this conversion is determined solely by the rate of reduction of 3'-dANO (3'-dANO reductase activity) and the ratio between the activities of adenosine kinase and adenosine deaminase in the cell. Consequently, the estimation of these enzyme activities in cell lysate of a given tumor can be used to predict whether the tumor is susceptible to inhibition by 3'-dANO.

Adenosine Deaminase↗
PubMed1981

Differential effect of N-carboxymethylisatoylation on the DNA polymerase activity, the 5' leads to 3'-exonuclease activity and the 3' leads to 5'-exonuclease activity of DNA polymerase I of Escherichia coli.

Treatment with native DNA polymerase I of Escherichia coli with the acylating agent N-carboxymethylisatoic acid anhydride (NCMIA) results under specific conditions in a rapid loss of polymerase activity, an increase in 5' leads to 3'-exonuclease activity and in unchanged 3' leads to 5'-exonuclease activity. When a nucleoside triphosphate and Mg2+ was present the polymerase activity was completely protected against the effect of NCMIA. Treatment with higher concentration of the acylating agent under these conditions led to a loss of 3' leads to 5'-exonuclease activity without any appreciable loss of polymerase activity. Treatment with NCMIA of the two catalytically active fragments of the enzyme led to very similar results. In this case both the polymerase activity and the 3' leads to 5'-exonuclease activity deteriorated more rapidly on treatment with the acylating reagent. The increase in 5' leads to 3'-exonuclease activity as a result of modification of the native enzyme appeared to be due to a change in the optimum conditions with regard to concentration of the assay buffer used. These changes are very similar to those seen when the polymerase is cleaved by limited proteolysis. From the results obtained it is concluded that NCMIA reacts primarily with a site at or near the triphosphate-Mg2+ complex binding site, leading to an almost complete loss of polymerase activity. The acylating reagent reacts also with another group on the native enzyme resulting in a modification of the 5' leads to 3'-exonuclease activity, and at high concentrations with a group leading to a slow loss of 3' leads to 5'-exonuclease activity.

Acylation↗