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Biomedical subjects

L F Liu

Publications and source records attributed to L F Liu.

At least 37 records · Page 2Linked to original sources
PubMed2000

Topoisomerase I inhibition and cytotoxicity of 5-bromo- and 5-phenylterbenzimidazoles.

Topoisomerase I is an enzyme that is essential for maintaining the three-dimensional structure of DNA during the processes of transcription, translation and mitosis. With the introduction of new clinical agents that are effective in poisoning topoisomerase I, this enzyme has proved to be an attractive molecular target in the development of anticancer drugs. Several terbenzimidazoles have been identified as potent topoisomerase I poisons. Structure-activity data on various terbenzimidazoles have revealed that the presence of lipophilic substituents at the 5-position of various terbenzimidazoles correlates with enhanced cytotoxicity. While the effect of having substituents at both the 5- and 6-positions had not been evaluated, previous studies did indicate that the presence of a fused benzo-ring at the 5,6-position results in a significant decrease in topoisomerase I poisoning activity and cytotoxicity. In the present study we investigated whether substituents at both the 5- and 6-positions of varied terbenzimidazoles would allow for retention of topo I poisoning activity. The 6-bromo, 6-methoxy, or 6-phenyl derivatives of both 5-bromo- and 5-phenylterbenzimidazole were synthesized and evaluated for topo I poisoning activity, as well as their cytotoxicity toward human lymphoblastoma cells. The data indicate that such derivatives do retain similar topo I poisoning activity and possess cytotoxicity equivalent to either 5-bromo- or 5-phenylterbenzimidazole. Significant enhancement in the topoisomerase I poisoning activity and cytotoxicity of 5-phenylterbenzimidazole is observed when the 2"-position is substituted with either a chloro or trifluoromethyl substituent. The influence of such substituents on the biological activity of 5.6-dibromoterbenzimidazole (6a) was also explored. In the case of either 2"-chloro-5,6-dibromoterbenzimidazole (6b) or 2"-trifluoromethyl-5,6-dibromoterbenzimidazole (6c), topoisomerase I poisoning was not enhanced relative to 6a. While cytotoxicity toward RPMI 8402 was also not significantly affected, comparative studies performed against several solid human tumor cell lines did reveal a significant increase in cytotoxicity observed for 6c as compared to 6a.

Benzimidazoles↗
PubMed2000

Mechanism of action of camptothecin.

Camptothecin (CPT) class of compounds has been demonstrated to be effective against a broad spectrum of tumors. Their molecular target has been firmly established to be human DNA topoisomerase I (topo I). CPT inhibits topo I by blocking the rejoining step of the cleavage/religation reaction of topo-I, resulting in accumulation of a covalent reaction intermediate, the cleavable complex. The primary mechanism of cell killing by CPT is S-phase-specific killing through potentially lethal collisions between advancing replication forks and topo-I cleavable complexes. Collisions with the transcription machinery have also been shown to trigger the formation of long-lived covalent topo-I DNA complexes, which contribute to CPT cytotoxicity. Two novel repair responses to topo-I-mediated DNA damage involving covalent modifications of topo-I have been discovered. The first involves activation of the ubiquitin/26S proteasome pathway, leading to degradation of topo-I (CPT-induced topo-I downregulation). The second involves SUMO conjugation to topo-I. The potentials roles of these new mechanisms for repair of topo-I-mediated DNA damage in determining CPT sensitivity/resistance in tumor cells are discussed.

Animals↗
PubMed1999

Characterization and cloning of avian-hepatic glutathione S-transferases.

Cytosolic glutathione S-transferases (GSTs) were isolated from 1-day-old Leghorn chick livers by glutathione (GSH)-affinity chromatography. After sample loading and extensive washing with 0.2 M NaCl, the column was sequentially eluted with 5 mM GSH and 1 mM S-hexylglutathione. The isolated GSTs were subjected to reverse-phase HPLC, electrospray ionization-MS, N-terminal and internal peptide sequencing analyses. The proteins recovered from the 5 mM GSH eluant were predominantly cGSTM1. A protein (cGSTM1') with an N-terminal amino acid sequence identical to that of cGSTM1 but with the initiator methionine retained and a novel class-mu isozyme (cGSTM2*) were also recovered from this fraction. Nine class-alpha isozymes with distinctive molecular masses were identified from the 1 mM S-hexylglutathione eluant. Three of these proteins are probably variants with minor amino acid substitutions of other isozymes. Of the six remaining class-alpha isozymes, three of them have had their complete (cGSTA1 and cGSTA2) or partial (cGSTA3) cDNA sequences reported previously in the literature. A chicken liver cDNA library was screened with oligonucleotides generated from the cGSTA2 sequence as probes. Clones that encompass the complete coding regions of cGSTA3 and cGSTA4 were obtained. A clone encoding the C-terminal 187 residues of cGSTA5 was also isolated.

Amino Acid Sequence↗
PubMed1999

Mutations of human topoisomerase II alpha affecting multidrug resistance and sensitivity.

Two mutations, R450Q and P803S, in the coding region of the human topoisomerase II alpha gene have been identified in the atypical multidrug resistant (at-MDR) cell line, CEM/VM-1, which exhibits resistance to many structurally diverse topoisomerase II-targeting antitumor drugs such as VM-26, doxorubicin, m-AMSA, and mitoxantrone. The R450Q mutation mapped in the ATP utilization domain, while the P803S mutation mapped in the vicinity of the active site tyrosine of human topoisomerase II alpha. However, the roles of these two mutations in conferring multidrug resistance are unclear. To study the roles of these two mutations in conferring multidrug resistance, we have characterized the recombinant human DNA topoisomerase II alpha containing either single or double mutations. We show that both R450Q and P803S mutations confer resistance in the absence of ATP. However, in the presence of ATP, the R450Q, but not the P803S, mutation can confer multidrug resistance. The R450Q enzyme was shown to exhibit impaired ATP utilization both for enzyme catalysis and for its ability to form the circular protein clamp. Interestingly, an unrelated mutation, G437E, which is also located in the same domain as the R450Q mutation, exhibited multidrug hypersensitivity in the absence of ATP. However, in the presence of ATP, the G437E enzyme is only minimally hypersensitive to various topoisomerase II drugs. In contrast to the R450Q enzyme, the G437E enzyme exhibited enhanced ATP utilization for enzyme catalysis. In the aggregate, these results support the notion that the multidrug resistance and sensitivity of these mutant enzymes are due to a specific defect in ATP utilization during enzyme catalysis.

Adenosine Triphosphate↗
PubMed1999

Plasmid linking number change induced by topoisomerase I-mediated DNA damage.

The state of cellular chromatin in response to DNA damage has been examined by monitoring the change in the linking number of circular episomes. COS cells transfected with an SV40-based vector were treated with camptothecin (CPT), a eukaryotic DNA topoisomerase I (TOP1) poison which induces TOP1-mediated DNA damage. Within minutes, a large increase in the linking number (over 10 linking number) of a small fraction (5-15%) of the episomal DNA was observed. A similar CPT-induced increase in plasmid DNA linking number was observed in Saccharomyces cerevisae expressing human DNA TOP1. In this case, the majority of the plasmid DNA can undergo rapid relaxation. The large increase in the plasmid linking number suggests major chromatin structural reorganization in response to TOP I-mediated DNA damage.

Animals↗
PubMed1999

Interaction between human topoisomerase I and a novel RING finger/arginine-serine protein.

The N-terminus of human topoisomerase I participates in the binding of this enzyme to helicases and other proteins. Using the N-terminal 250 amino acids of human topoisomerase I and a yeast two-hybrid/ in vitro binding screen, a novel arginine-serine-rich peptide was identified as a human topoisomerase I-binding protein. The corresponding full-length protein, named topors, contains a consensus RING zinc finger domain and nuclear localization signals in addition to the arginine-serine-rich region. The RING finger domain of topors is homologous to a similar domain in a family of viral proteins that are involved in the regulation of viral transcription. When expressed in HeLa cells as a green fluorescent protein fusion, topors localizes in the nucleus in a punctate pattern and co-immunoprecipitates with topoisomerase I. These data suggest that topors is involved in trans-cription, possibly recruiting topoisomerase I to RNA polymerase II transcriptional complexes.

Amino Acid Sequence↗
PubMed1999

Activation of topoisomerase II-mediated excision of chromosomal DNA loops during oxidative stress.

Hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is known to induce oxidative stress and apoptosis. U937 cells treated with H2O2 were shown to produce high molecular weight (HMW) DNA fragments approximately 50 to 100 kb in size in <1 min. The formation of these HMW DNA fragments is reversible and shown to be mediated by DNA topoisomerase II (TOP2). Following this initial event, formation of irreversible HMW DNA fragments and nucleosomal ladders occurs. Our results thus demonstrate a potential role of TOP2 in oxidative damage of DNA and apoptotic cell death.

DNA Damage↗
PubMed1999

Co-expression of glutathione S-transferase with methionine aminopeptidase: a system of producing enriched N-terminal processed proteins in Escherichia coli.

We describe here an Escherichia coli expression system that produces recombinant proteins enriched in the N-terminal processed form, by using glutathione S-transferase cGSTM1-1 and rGSTT1-1 as models, where c and r refer to chick and rat respectively. Approximately 90% of the cGSTM1-1 or rGSTT1-1 overexpressed in E. coli under the control of a phoA promoter retained the initiator methionine residue that was absent from the mature isoenzymes isolated from tissues. The amount of initiator methionine was decreased to 40% of the expressed cGSTM1-1 when the isoenzyme was co-expressed with an exogenous methionine aminopeptidase gene under the control of a separate phoA promoter. The recombinant proteins expressed were mainly methionine aminopeptidase. The yield of cGSTM1-1 was decreased to 10% of that expressed in the absence of the exogenous methionine aminopeptidase gene. By replacing the phoA with its natural promoter, the expression of methionine aminopeptidase decreased drastically. The yield of the co-expressed cGSTM1-1 was approx. 60% of that in the absence of the exogenous methionine aminopeptidase gene; approx. 65% of the initiator methionine residues were removed from the enzyme. Under similar conditions, N-terminal processing was observed in approx. 70% of the recombinant rGSTT1-1 expressed. By increasing the concentration of phosphate in the growth medium, the amount of initiator methionine on cGSTM1-1 was decreased to 14% of the overexpressed isoenzymes, whereas no further improvement could be observed for rGSTT1-1. The initiator methionine residue does not affect the enzymic activities of either cGSTM1-1 or rGSTT1-1. However, the epoxidase activity and the 4-nitrobenzyl chloride-conjugating activity of the purified recombinant rGSTT1-1 are markedly higher that those reported recently for the same isoenzyme isolated from rat livers.

Aminopeptidases↗
PubMed1999

Design of antineoplastic agents based on the "2-phenylnaphthalene-type" structural pattern. 4. Synthesis and biological activity of 2-chloro-3-(substituted phenoxy)-1, 4-naphthoquinones and related 5,8-dihydroxy-1,4-naphthoquinones.

The intermediate in the preparation of 1,3,7, 10-tetrahydroxybenzo[b]naphtho[2,3-d]furan-6,11-dione (2), 2-chloro-5,8-dimethoxy-3-(3,5-dimethoxyphenoxy)-1,4-naphthoquinone (8h), and corresponding hydroxyl, methoxyl, and acetoxyl analogues was found to possess interesting inhibitory activities in a number of cytotoxic test systems. Activities were also noticed in some 5, 8-dihydroxy-1,4-naphthoquinone derivatives. A structure-activity discussion of compounds of this series is presented. The newly uncovered biological activity of 2-chloro-3-(substituted phenoxyl)-1, 4-naphthoquinones and 2,3-bis(substituted phenoxy)-1, 4-naphthoquinones may suggest an approach for the development of new classes of antineoplastic agents.

Antineoplastic Agents↗
PubMed1999

Inversion/dimerization of plasmids mediated by inverted repeats.

In contrast with earlier studies on the lambda and Escherichia coli genomes, recombination between inverted repeats on plasmids is highly efficient and shown to be recA-independent. In addition, the recombination product is exclusively a head-to-head inverted dimer. Here, we show that this recombination/rearrangement event can occur on different plasmid replicons and is not specific to the particular sequence within the inverted repeats. Transcription readthrough into the inverted repeats has little effect on this event. Genetic analysis has also indicated that most known recombination enzymes are not involved in this process. Specifically, single or double mutants defective in Holliday junction resolution systems (RuvABC and/or RecG/RusA) do not abolish this recombination/rearrangement event. This result does not support the previous models (i.e. the reciprocal-strand-switching and the cruciform-dumbbell models) in which intermediates containing Holliday junctions are proposed. Further analysis has demonstrated that the recombination/rearrangement frequency is dramatically (over 1000-fold) reduced if mismatches (2.8 %) are present within the inverted repeats. Mutations in dam, mutH and mutL genes partially or completely restored the recombination/rearrangement frequency to the level exhibited by the perfect inverted repeats, suggesting the formation of heteroduplexes during recombination/rearrangement. Sequencing analysis of the recombination/rearrangement products have indicated that the majority of the products do not involve crossing-over. We discuss a possible mechanism in which blockage of the lagging strand polymerase by a hairpin triggers recombination/rearrangement mediated by inverted repeats.

Base Sequence↗
PubMed1999

Differentially and developmentally regulated expression of three rice sucrose synthase genes.

The spatial and temporal distribution of sucrose synthase (RSuS) in rice (Oryza sativa L.) was studied by Western and immunohistochemical analyses using the monospecific antibodies for three RSuS isoforms. In leaf tissues, RSuS1 was localized in the mesophyll while RSuS2 was in the phloem in addition to the mesophyll. In the roots, only RSuS1 was found in the phloem. No RSuS3 could be detected in any parts of etiolated seedlings. The expression of each RSus gene is closely linked to the seed development. RSuS1 was present in the aleurone layers of developing seeds, and at a low level in endosperm cells. RSuS2 was evenly distributed in seed tissues other than the endosperm. RSuS3 was localized predominantly in the endosperm cells. The tissue specific localizations of the three gene products suggest that RSuS1 plays a role in sugar transport into endosperm cells where the reaction catalyzed by RSuS3 provides the precursor of starch synthesis. RSus2, which is ubiquitously expressed, may play a housekeeping role.

Amino Acid Sequence↗
PubMed1998

Genetic characterization of the mRNAs encoding alpha-bungarotoxin: isoforms and RNA editing in Bungarus multicinctus gland cells.

The mRNA encoding alpha-bungarotoxin (alpha-Butx) was prepared from the venom glands of Bungarus multicinctus by Cap-finder cDNA synthesis. The sequences of the 3'- and 5'-flanking regions including a signal peptide of alpha-Butx were almost identical with those of Elapidae and Hydrophiidae toxins, suggesting that they may have the same origin. Sixteen polymorphic mRNA sequences of alpha-Butx were detected from B.multicinctus gland cells. Analysis of the genomic DNA of alpha-Butx indicated that the polymorphic mRNA originated from one DNA sequence. Most of the mutations in alpha-Butx mRNA were silent and the hot-spot variations occurred at 78, 107, 129, 198 and 201 nt in alpha-Butx mRNA. Ten distinct protein sequences of alpha-Butx could be deduced from the polymorphic mRNA and one of the isoforms has already been isolated. Since alpha-Butx DNA is a single copy in the genome, the RNA polymorphism might result from post-transcriptional editing. These results indicate that the authentic alpha-Butx is in fact derived from edited mRNAs. RNA editing may contribute a common mechanism toward the diversity of alpha-neurotoxins in snake glands.

Amino Acid Sequence↗
PubMed1998

Selective cytotoxicity of topoisomerase-directed protoberberines against glioblastoma cells.

Protoberberines are a new class of organic cations that are dual poisons of topoisomerases I and II. Certain protoberberines exhibit greater in vitro cytotoxicity against cell lines derived from solid tumors than from leukemias. Using a group of seventeen different protoberberine analogs, the structural basis for selective cytotoxicity toward sensitive SF-268 glioblastoma cells as compared with resistant RPMI 8402 lymphoblast cells was explored. The selective cytotoxicity is associated with the presence of an imminium ion and other structural features of protoberberines, and is not shared by drugs such as camptothecin, doxorubicin, vinblastine, and etoposide, which are either equally or more cytotoxic against RPMI 8402 cells than SF-268 cells. The selective cytotoxicity of protoberberines against SF-268 over RPMI 8402 cells is not due to differences in topoisomerase levels or known drug efflux systems such as multidrug resistance (MDR1) and multidrug-resistance protein (MRP). Comparative in vitro studies of the accumulation of coralyne, a fluorescent protoberberine, into sensitive and resistant cells demonstrated a correlation between drug accumulation and selective cytotoxicity. Inhibitors of coralyne uptake included several protoberberine-related compounds. Of these, palmatine, a minimally cytotoxic protoberberine, both inhibited coralyne accumulation and reduced cytotoxicity against SF-268 cells, but not against RPMI 8402 cells. Despite the structural resemblance of protoberberines to catecholamines, our experiments using inhibitors and cells expressing biogenic amine uptake systems have ruled out the involvement of biogenic amine uptake1, uptake2, and vesicular monoamine transport systems. Uptake systems remaining as candidates, supported by preliminary data, include transport via vesicles derived from specialized membrane invaginations and selected carrier-mediated organic amine transport systems.

Antineoplastic Agents↗
PubMed1998

DNA minor groove binding-directed poisoning of human DNA topoisomerase I by terbenzimidazoles.

We have employed a broad range of spectroscopic, calorimetric, DNA cleavage, and DNA winding/unwinding measurements to characterize the DNA binding and topoisomerase I (TOP1) poisoning properties of three terbenzimidazole analogues, 5-phenylterbenzimidazole (5PTB), terbenzimidazole (TB), and 5-(naphthyl[2,3-d]imidazo-2-yl)bibenzimidazole (5NIBB), which differ with respect to the substitutions at their C5 and/or C6 positions. Our results reveal the following significant features. (i) The overall extent to which the three terbenzimidazole analogues poison human TOP1 follows the hierarchy 5PTB > TB >> 5NIBB. (ii) The impact of the three terbenzimidazole analogues on the superhelical state of plasmid DNA depends on the [total ligand] to [base pair] ratio (rbp), having no effect on DNA superhelicity at rbp ratios < or = 0.1, while weakly unwinding DNA at rbp ratios > 0.1. This weak DNA unwinding activity exhibited by the three terbenzimidazoles does not appear to be correlated with the abilities of these compounds to poison TOP1. (iii) Upon complexation with both poly(dA).poly(dT) and salmon testes DNA, the three terbenzimidazole analogues exhibit flow linear dichroism properties characteristic of a minor groove-directed mode of binding to these host DNA duplexes. (iv) The apparent minor groove binding affinities of the three terbenzimidazole analogues for the d(GA4T4C)2 duplex follow a qualitatively similar hierarchy to that noted above for ligand-induced poisoning of human TOP1-namely, 5PTB > TB > 5NIBB. In the aggregate, our results suggest that DNA minor groove binding, but not DNA unwinding, is important in the poisoning of TOP1 by terbenzimidazoles.

Animals↗
PubMed1998

Quantitative structure-activity relationships on 5-substituted terbenzimidazoles as topoisomerase I poisons and antitumor agents.

Several 5-substituted terbenzimidazoles were synthesized and evaluated as mammalian topoisomerase I poisons and for cytotoxicity against a human lymphoblastoma cell line, RPMI-8402. No correlation was observed between topoisomerase I poisoning activity and the Hansch pi value or the sigma meta and sigma para values associated with each substituent. These data suggest that electronic effects and relative lipophilicity of substituents at the 5-position of these terbenzimidazoles do not have a significant effect upon intrinsic topoisomerase I poisoning activity. There was, however, a good correlation between the relative pi values for the various substituents evaluated and cytotoxic activity. Experimentally determined log P values did not correlate well with either cytotoxicity or pi values. Capacity factors (log k') as determined by high pressure liquid chromatography did correlate well with the pi values of varied substituents and cytotoxicity. These data indicate that the relative lipophilic activity of substituents at the 5-position of these terbenzimidazoles can strongly influence relative cytotoxic activity.

Antineoplastic Agents↗
PubMed1998

Modulation of gyrase-mediated DNA cleavage and cell killing by ATP.

An uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, and an aconitase inhibitor, fluoroacetic acid, both of which are known to lower the cellular ATP pool, protected Escherichia coli cells from the bactericidal actions of gyrase poisons including quinolone antibiotics, nalidixic acid and ciprofloxacin, and the epipodophyllotoxins VP-16 and VM-26. Using purified E. coli DNA gyrase, we examined the effect of ATP on gyrase-mediated DNA cleavage in the presence of these gyrase poisons. ATP was shown to stimulate gyrase-mediated DNA cleavage from 10- to more than 100-fold in the presence of these gyrase poisons. ADP antagonized the stimulatory effect of ATP. Consequently, gyrase-mediated DNA cleavage induced by gyrase poisons is modulated by the ATP concentration/ADP concentration ([ATP]/[ADP]) ratio. Coumermycin A1, an inhibitor of the ATPase subunit of DNA gyrase, like ADP, also effectively antagonized the stimulatory effect of ATP on gyrase-mediated DNA cleavage induced by gyrase poisons. Furthermore, coumermycin A1, like DNP and fluoroacetic acid, also protected cells from the bactericidal action of gyrase poisons. In the aggregate, our results are consistent with the notion that the [ATP]/[ADP] ratio, through its modulatory effect on the gyrase-mediated DNA cleavage, is an important determinant of cellular susceptibility to gyrase poisons.

Adenosine Diphosphate↗