Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Enhanced DNA repair has been identified as a major mechanism of resistance to the anticancer drug cisplatin in murine leukemia L1210 cells. Studies of other cells have implicated the elevation of a variety of RNA transcripts in cisplatin resistance. This study investigated potential changes in transcription of these genes as well as genes involved in DNA repair. No elevation in any of the following transcripts was observed: thymidylate synthase, dihydrofolate reductase, DNA polymerase alpha, DNA polymerase beta, topoisomerase II, Ha-ras, beta-tubulin, metallothionein and the DNA repair genes ERCC1 and ERCC2. Thymidine kinase was increased no more than 2-fold. None of these RNA were induced by incubation with cisplatin. High levels of cisplatin produced selective decreases in certain RNA. These results demonstrate that the previous observations of elevated RNA can not be universally applied to all cisplatin-resistant cells.
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PMID:Analysis of various mRNA potentially involved in cisplatin resistance of murine leukemia L1210 cells. 197 66

The enzymology of DNA repair is currently under active investigation. The purpose of the present study was to examine the involvement of a number of enzymes (DNA polymerase alpha and beta, DNA topoisomerase II and ribonucleotide reductase) in the repair of chemically induced DNA damage in a mammalian cell system. This was done by studying the effects of inhibitors of these enzymes on the levels of 2-acetylaminofluorene (2-AAF)-DNA adducts and on the induction of UDS in primary cultures of rat hepatocytes exposed to the carcinogen in vitro. The results obtained with aphidicolin (an inhibitor of DNA polymerase alpha) show that the binding of 2-AAF to cellular DNA was significantly higher in samples exposed to this compound. Moreover, induction of UDS by 2-AAF was completely blocked in the presence of this compound. Dideoxythymidine, a DNA polymerase beta inhibitor, led to complex results. It produced a reduced DNA-specific activity due to [3H]2-AAF adduct formation as well as a diminished but still detectable UDS response in the presence of 2-AAF. Inhibitors of DNA topoisomerase II (nalidixic acid) and ribonucleotide reductase (hydroxyurea) did not cause any statistically significant change in the accumulation of 2-AAF adducts nor did they affect the induction of UDS. The data clearly suggest that DNA polymerase alpha participates in the repair of 2-AAF adducts in hepatocytes. In addition, neither DNA topoisomerase II activity, nor limitations in the precursor nucleotide pools appear to be critical factors in this process.
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PMID:The effects of putative DNA repair inhibitors on DNA adduct levels and unscheduled DNA synthesis in rat hepatocytes exposed to 2-acetylaminofluorene. 253 61

The inhibition of the semiconservative and restorative DNA synthesis caused by hyperthermia (30 to 60 min, 43 degrees C) was significantly higher in spleen cells than in thymus cells. The DNA repair synthesis of thymus cells measured at 37 degrees C was increased by about two times the initial value after a pre-incubation of 30 to 90 min and 30 to 60 min, respectively, with 37 and 43 degrees C, respectively. Under the same conditions, the 3H-thymidine incorporation into the DNA of spleen cells diminished proportionally to the pre-incubation time after a pre-incubation of 30 and 45 min, respectively, with 43 and 37 degrees C, respectively. When hyperthermia and inhibitors of DNA synthesis or DNA repair (hydroxyurea, 1-beta-D-arabinofuranosylcytosine, 3',5'-didesoxythymidine, and 3-aminobenzamide) were combined, overadditive effects--without cell specific particularities--were seen only in the case of 3-aminobenzamide. Only in thymus cells, the inhibitor of DNA topoisomerase II novobiocin caused an overadditive reinforcement of the inhibition induced by hyperthermia of the semiconservative DNA synthesis. The stimulation of DNA repair synthesis in thymus cells caused by novobiocin with the aid of DNA polymerase beta could be compensated by hyperthermia. The sedimentation of thymus and spleen cell nucleoids was increased after hyperthermia. The results suggest a special importance of DNA topology and of the DNA polymerase beta activity for the cellular effect of hyperthermia.
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PMID:[Deoxyribonucleic acid synthesis by rat thymus and spleen cells in vitro following hyperthermia]. 283 58

Administration of hepatocarcinogens aflataxin B1 (AFB1) and N-nitrosodimethylamine (NDMA) to rats caused single-strand breaks in hepatic nuclear DNA. The damage was found to be maximum at 4 hours following AFB1 administration and at 2 hours following NDMA administration. These damages were repaired after 17 and 4 hours, respectively in cases of AFB1 and NDMA. The activity of poly(ADP-ribose)polymerase (PARP), an enzyme known to use single-strand breaks of DNA as cofactor, was observed to increase with increasing damage to DNA and decrease as and when this damage got repaired. DNA polymerase beta and DNA ligase activities were also seen to increase and decline in a way analogous to PARP. In contrast, DNA topoisomerase activity declined corresponding to an increase in PARP activity. These observations suggest a possible role of PARP in coordinating the activities of other enzymes involved in DNA repair. It is also envisaged that these parameters can be utilized to devise strategies to counteract the deleterious effects of chemical carcinogens.
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PMID:Activity of some nuclear enzymes associated with DNA repair following hepatocarcinogen administration to rats. 759 30

Previous investigations have revealed that the human TE-671 MR human rhabdomyosarcoma xenograft selected in vivo for melphalan resistance (M. C. Rosenberg, et al., Cancer Res., 49: 6917-6922, 1989) is cross-resistant to a wide variety of alkylating agents and to bleomycin, but is collaterally sensitive to etoposide. Although glutathione levels were noted to be elevated in TE-671 MR compared to the melphalan-sensitive parental TE-671 xenograft, treatment with buthionine sulfoximine to deplete glutathione levels did not fully restore melphalan sensitivity in the TE-671 MR xenograft. The present studies were undertaken to search for additional mechanisms of resistance in the TE-671 MR xenograft. Drug sensitivity testing performed at the dose of agents that was lethal to 10% of the animals revealed that the TE-671 MR xenograft maintained resistance to the bifunctional cross-linking agent 1,3-bis(2-chloroethyl)-1-nitrosourea and was cross-resistant to the topoisomerase I poison topotecan. Treatment with buthionine sulfoximine did not sensitize the TE-671 MR xenograft to 1,3-bis(2-chloroethyl)-1-nitrosourea. Further, even though O6-alkylguanine-DNA alkyltransferase levels were high in both the TE-671 and TE-671 MR xenografts, depletion of O6-alkylguanine-DNA alkyltransferase activity by treatment with O6-benzylguanine substantially sensitized the TE-671 xenografts but not the TE-671 MR xenografts, suggesting an additional mechanism of resistance. Measurement of additional enzyme activities that might be involved in DNA repair revealed significant elevations in DNA polymerase alpha (46 +/- 8 (SD) units/mg protein in TE-671, 69 +/- 6 units/mg protein in TE-671 MR, P < 0.05) and DNA polymerase beta (0.43 +/- 0.01 units/mg protein in TE-671, 0.78 +/- 0.12 units/mg protein in TE-671 MR, P < 0.05) but not DNA polymerase delta or total DNA ligase. Examination of topoisomerases by activity assays and Western blotting revealed a 2-fold increase in topoisomerase II and a 2-fold decrease in topoisomerase I in the TE-671 MR xenograft compared to the parental xenograft, apparently explaining the collateral sensitivity to etoposide and cross-resistance to topotecan. These results suggest that TE-671 MR xenografts contain multiple changes in activities of DNA repair-related proteins and other nuclear proteins that could contribute to alkylating agent resistance.
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PMID:Elevated DNA polymerase alpha, DNA polymerase beta, and DNA topoisomerase II in a melphalan-resistant rhabdomyosarcoma xenograft that is cross-resistant to nitrosoureas and topotecan. 801 71

We have purified to near homogeneity a DNA primase from a mitochondrial fraction of the trypanosomatid Crithidia fasciculata. The enzyme is a single polypeptide chain of 28 kDa. Using a poly(dT) template and ATP as a substrate, the enzyme makes oligonucleotides of which the vast majority are about 10 nucleotides in size or smaller. With a single-stranded M13 DNA template and the four rNTPs as substrates, the enzyme makes heterogeneous oligonucleotides in the same size range. These oligonucleotides efficiently prime the synthesis of DNA by the Klenow DNA polymerase. Immunolocalization with antibodies against the purified enzyme confirms that the primase is mitochondrial. Furthermore, the enzyme localizes to specific regions of the cell's single mitochondrion, above and below the condensed kinetoplast DNA. The primase does not co-localize with the mitochondrial topoisomerase II and DNA polymerase beta, both of which are associated with two protein complexes positioned on opposite sides of the kinetoplast disc. These localization studies have significant implications for the mechanism of kinetoplast DNA replication.
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PMID:A mitochondrial DNA primase from the trypanosomatid Crithidia fasciculata. 925 2

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is present in tobacco smoke and is hepatocarcinogenic in rats. Its bioactivation in rat hepatocytes leads to methylation and pyridyloxobutylation of DNA. Rat hepatocytes were cultured in serum-free William medium E on collagen-coated dishes. We demonstrated that some enzymes of the base and/or excision-repair pathways were involved in repair of NNK-induced DNA damage, measured by [methyl-3H] thymidine incorporation. Unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU), NNK, N'-nitrosonornicotine (NNN) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) increased 2.9-, 2.8-, 1.5- and 3.5-fold, respectively, suggesting that methylated and/or pyridyloxobutylated-DNA by these four nitroso compounds is repaired by the excision pathway. Moreover, levels of NNK-induced UDS were dose (1-3 mM) and time (1-18 h) dependent. Enzymes involved in the excision repair pathways were selectively inhibited. Inhibitors of DNA topoisomerase I (camptothecin) and topoisomerase II (etoposide, nalidixic acid) did not decrease the induction of UDS, suggesting that topoisomerases are not involved in the repair of NNK-induced damage. While aphidicolin and arabinocytidine (DNA polymerase alpha, delta, epsilon inhibitors) totally inhibited NNK- and NNKOAc-induced UDS, dideoxythymidine (DNA polymerase beta inhibitor) inhibited NNK- and NNKOAc-induced UDS by 40 and 33%, respectively. We conclude that DNA polymerase alpha, delta or epsilon and to a lesser degree polymerase beta are involved in the repair of pyridyloxobutylated DNA. Previous studies showed that inhibition of poly(ADP-ribosyl) polymerase (PARP) by 3-aminobenzamide (3-ab) facilitated DNA ligation. Our results demonstrate that 3-ab increased NNK-induced UDS, but does not affect NNKOAc-induced UDS. These observations suggest that the ligation step is rate limiting in the repair of methylated DNA but not of pyridyloxobutylated DNA.
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PMID:Modulation of DNA repair by various inhibitors of DNA synthesis following 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced DNA damage. 956 22

Kinetoplast DNA (kDNA), the mitochondrial DNA in kinetoplastids, is a network containing several thousand topologically interlocked minicircles. We investigated cell cycle-dependent changes in the localization of kDNA replication enzymes by combining immunofluorescence with either hydroxyurea synchronization or incorporation of fluorescein-dUTP into the endogenous gaps of newly replicated minicircles. We found that while both topoisomerase II and DNA polymerase beta colocalize in two antipodal sites flanking the kDNA during replication, they behave differently at other times. Polymerase beta is not detected by immunofluorescence either during cell division or G1, but is abruptly detected in the antipodal sites at the onset of kDNA replication. In contrast, topoisomerase II is localized to sites at the network edge at all cell cycle stages; usually it is found in two antipodal sites, but during cytokinesis each postscission daughter network is associated with only a single site. During the subsequent G1, topoisomerase accumulates in a second localization site, forming the characteristic antipodal pattern. These data suggest that these sites at the network periphery are permanent components of the mitochondrial architecture that function in kDNA replication.
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PMID:Changes in organization of Crithidia fasciculata kinetoplast DNA replication proteins during the cell cycle. 981 50

The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata has an unusual structure composed of minicircles and maxicircles topologically interlocked into a single network and organized in a disc-shaped structure at the base of the flagellum. We previously purified a structure-specific endonuclease (SSE1), based on its RNase H activity, that is enriched in isolated kinetoplasts. The endonuclease gene has now been cloned, sequenced, and found to be closely related to the 5' exonuclease domain of bacterial DNA polymerase I proteins. Although the protein does not contain a typical mitochondrial leader sequence, the enzyme is shown to colocalize with a type II DNA topoisomerase and a DNA polymerase beta at antipodal sites flanking the kinetoplast disc. Cell synchronization studies with an epitope-tagged construct show that the localization of the endonuclease to the antipodal sites varies in a cell cycle-dependent manner similar to that of the DNA polymerase beta [Johnson, C. E. & Englund, P. T. (1998) J. Cell Biol. 143, 911-919]. Immunofluorescent localization of SSE1 to the antipodal sites is only observed during kinetoplast replication. Together, these results suggest a point of control for kinetoplast DNA replication through the regulation of the availability of DNA replication proteins and a possible role for the antipodal sites in removal of RNA primers and the repair of gaps in newly replicated minicircles.
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PMID:The kinetoplast structure-specific endonuclease I is related to the 5' exo/endonuclease domain of bacterial DNA polymerase I and colocalizes with the kinetoplast topoisomerase II and DNA polymerase beta during replication. 1041 96

Previous studies have demonstrated alkylating (melphalan) resistance in the B-CLL derived WSU-CLL cell line as compared to WIL2 B lymphocytic cells. Nuclear extracts from WSU-CLL cells demonstrate a highly significant increase in DNA topoisomerase II activity as compared to WIL2 cells. Western blot analysis showed the level of topoisomerase II proteins expressed in WSU-CLL cells to be increased as compared to WIL2 cells. WSU-CLL cells were 5.24-fold more sensitive than WIL2 cells to the cytotoxic effect of the topoisomerase II inhibitor doxorubicin. No difference in topoisomerase I activity or of the level of topoisomerase I protein expression was observed comparing the two cell lines. The sensitivity to the cytotoxic effects of topoisomerase I inhibitor, camptothecin, did not differ in WSU-CLL and WIL2 cell lines. Pre-incubation with doxorubicin significantly increased melphalan induced interstrand-DNA-crosslink formation and cytotoxicity in WSU-CLL cells as compared to WIL2 cells. The affinity of topoisomerase II for WSU-CLL UV-irradiated-crosslinked DNA was increased 2.84-fold as compared to that of WSU-CLL native DNA. The affinity of topoisomerase II for both UV-irradiated (crosslinked) and for native DNA was significantly decreased after doxorubicin-pretreatment. Measurement of DNA polymerase beta and DNA polymerase beta revealed significant elevations in DNA polymerase beta (58.82 +/- 3.67 units/mg protein in WSU-CLL cells, as compared to 27.82 +/- 4.39 units/mg protein in WIL 2 cells; p < 0.01) but not DNA polymerase beta (0.82 +/- 0.11 units/mg protein in WSU-CLL cells, compared to 0.74 +/- 0.09 units/mg protein in WIL2, p > 0.05). However, exposure to aphidicolin (an inhibitor of DNA polymerase a) failed to increase melphalan induced cytotoxicity suggesting that although DNA polymerase a activity was increased in WSU-CLL cells the mechanisms of resistance does not involve this specific DNA repair pathway. Elevated topoisomerase II activity and the increased affinity of topoisomerase II for crosslinked DNA in melphalan resistant cells appears to be the major factor responsible for alkylator resistance by changing DNA topology and thereby facilitating DNA repair.
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PMID:Alkylator resistance in human B lymphoid cell lines: (2). Increased levels of topoisomerase II expression and function in a melphalan-resistant B-CLL cell line. 1095 28


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