Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have previously shown that blockade of the Na+,K(+)-pump by the cardiac glycoside ouabain produces doxorubicin resistance and decreases topoisomerase II-mediated DNA strand breakage in hamster cells. To determine if this were a general phenomenon, the effect of pump blockade on doxorubicin resistance was assessed in three human tumor cell lines: A549 lung and HT29 colon adenocarcinomas and U1 melanoma. When cells were exposed to 1 microM ouabain prior to and during incubation with doxorubicin, cytotoxicity was markedly reduced. Ouabain had no effect on either the influx or the efflux of doxorubicin. However, all cell lines showed a ouabain-induced decrease in doxorubicin-induced topoisomerase-mediated DNA strand breakage (SSB). These data suggest that blockade of the Na+,K+ pump decreases doxorubicin cytotoxicity in human tumor cells by inhibiting topoisomerase-mediated SSB. Furthermore, they indicate that altered ionic gradients are a potential cause of resistance to drugs that use topoisomerase II as a target.
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PMID:The influence of Na+,K(+)-pump blockade on doxorubicin-mediated cytotoxicity and DNA strand breakage in human tumor cells. 216 43

We have found that blockade of the Na+,K+-pump by the cardiac glycoside ouabain protects human A549 and hamster V79 cells from the cytotoxic effects of the topoisomerase II poison etoposide. One thousand-fold higher concentrations of ouabain were required to protect V79 cells compared to A549 cells. Since this difference parallels previously measured differences in pump sensitivity, it suggests that protection is mediated directly through pump blockade. Ouabain affected neither the cellular influx nor efflux of etoposide. However, pump blockade did decrease the formation of etoposide-induced DNA-topoisomerase, II-cleavable complexes, assessed as single and double strand DNA breaks using alkaline and neutral elution. To determine if this decrease were a direct effect of change in ionic environment produced by pump blockade, experiments with isolated nuclei and partially purified topoisomerase II were performed. Etoposide-induced cleavable complex formation and topoisomerase-mediated decatenation were assessed in buffers which mimicked either normal intracellular ionic conditions or those produced by ouabain. Compared to the buffer which resembled the normal intracellular ionic conditions, the buffer that mimicked the conditions produced by pump blockade produced fewer etoposide-mediated cleavable complexes in isolated nuclei and less decatenating activity of partially purified topoisomerase II. These findings demonstrate that inhibition of the Na+,K+-pump causes an alteration in the intracellular ionic environment which decreases the activity of topoisomerase II, thus producing a decrease in etoposide-induced cleavable complex formation and cytotoxicity. Since ionic changes occur inside normal cells during progression through the cell cycle as well as in cells that have undergone transformation, these data suggest that the intracellular ionic environment plays a role in determining the sensitivity of normal and malignant cells to this group of chemotherapeutic agents.
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PMID:Dependence of etoposide-induced cytotoxicity and topoisomerase II-mediated DNA strand breakage on the intracellular ionic environment. 254 16

The cardiac glycoside ouabain, which is a specific inhibitor of the Na+,K+-pump, confers dramatic protection from the cytotoxic effects of doxorubicin (Adriamycin). This effect was documented in cultured A549 cells (human lung adenocarcinoma). CCL210 cells (human fibroblasts), and V79 cells (hamster fibroblasts). Maximum protection from doxorubicin cytotoxicity was achieved using 1 microM ouabain for A549 and CCL210 cells and 300 microM ouabain for V79 cells. These concentrations correlated well with the concentrations of ouabain required to induce Na+,K+-pump blockade, which was assessed using the K+ analogue 86Rb+. This suggests that protection is mediated by pump blockade. Addition of ouabain at the same time as doxorubicin was just as protective as preincubation with ouabain for an hour, demonstrating that the ouabain acts rapidly. Ouabain treatment affected neither influx nor efflux of doxorubicin. Ouabain also had no effect on verapamil-induced inhibition of doxorubicin efflux. However, ouabain partially blocked the verapamil-induced potentiation of the cytotoxic effects of doxorubicin. Therefore, ouabain does not protect by affecting intracellular doxorubicin levels. Fluorescence microscopy showed that the ability of doxorubicin to reach the nucleus was not influenced by ouabain. Alkaline elution studies demonstrated that ouabain greatly decreased doxorubicin-induced DNA strand breakage. Protection from cytotoxicity correlated closely with this decrease in strand breakage. These studies suggest that the stabilization of DNA-topoisomerase II complexes is closely linked to the mechanism of doxorubicin cytotoxicity and that this stabilization is influenced by the intracellular ionic milieu.
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PMID:Reduction of doxorubicin cytotoxicity by ouabain: correlation with topoisomerase-induced DNA strand breakage in human and hamster cells. 282 82