Biomedical subjects

L F Liu

Publications and source records attributed to L F Liu.

At least 145 records · Page 8Linked to original sources

PubMed1988

Studies on the effect of tetrandrine on microtubules. II. Observation with immunofluorescence techniques.

Microtubules were purified by using two cycles of assembly and disassembly processes on fresh brain homogenates from 30 guinea pigs. The yield was about 60 mg. The effect of tetrandrine on tubulin was determined by spectrophotometric analysis and electron microscopy. In addition, we used the indirect immunofluorescent method including tubulin antibody to locate the presence of microtubules in 3T3 cells by fluorescence microscopy. The effects of colchicine and P204 were studied for comparison at the same time. The results showed that colchicine can effectively depolymerize microtubules, while tetrandrine showed aggregation, and in a different manner. The shape and structure of microtubules were definitely destroyed by colchicine, but were not affected by P204 which protected against the destructive effect of tetrandrine. This result indicated the safety of using a combination of P204 and tetrandrine in the treatment of silicosis.

Alkaloids↗

PubMed1988

Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22.

Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage lambda was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a lambda gt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes.

Base Sequence↗

PubMed1988

Human DNA topoisomerase I is encoded by a single-copy gene that maps to chromosome region 20q12-13.2.

cDNA clones of the human TOP1 gene encoding DNA topoisomerase I (EC 5.99.1.2) have been obtained by immunochemical screening of phage lambda libraries expressing human cDNA segments, using rabbit antibodies raised against purified HeLa DNA topoisomerase I. Hybridization patterns between the cloned cDNA sequences and human cellular DNA and cytoplasmic mRNAs indicate that human TOP1 is a single-copy gene. The chromosomal location of the gene has been mapped to the long arm of chromosome 20, in the region q12-13.2, by hybridization of a radioactively labeled TOP1 cDNA probe to human metaphase chromosomes and to a panel of rodent-human somatic hybrids retaining overlapping subsets of human chromosomes.

Animals↗

PubMed1988

Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes.

Treatment of HeLa cells with a DNA topoisomerase I-specific inhibitor, camptothecin, results in rapid cessation of the synthesis of the 45S rRNA precursor. The inhibition of rRNA synthesis is reversible following drug removal and correlates with the presence of camptothecin-trapped topoisomerase I-DNA abortive complexes, which can be detected as topoisomerase I-linked DNA breaks upon lysis with sodium dodecyl sulfate. These breaks were found to be concentrated within the transcribed region of human rRNA genes. No such sites can be detected in the inactive human rRNA genes in mouse-human hybrid cells, suggesting a preferential association of topoisomerase I with actively transcribed genes. The distribution of RNA polymerase molecules along the transcription unit of human rRNA genes in camptothecin-treated HeLa cells, as assayed by nuclear run-on transcription, shows a graded decrease of the RNA polymerase density toward the 3' end of the transcription unit; the density is minimally affected near the 5' start of the transcription unit. These results suggest that DNA topoisomerase I is normally involved in the elongation step of transcription, especially when the transcripts are long, and that camptothecin interferes with this role.

Camptothecin↗

PubMed1988

Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro.

We have found that purified calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus DNA topoisomerase II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoisomerase. The subunit exchange model, which has been suggested for the DNA gyrase-mediated recombination, is now generalized as follows: DNA topoisomerase II molecules bind to DNAs, associate with each other, and lead to the exchange of DNA strands through the exchange of topoisomerase II subunits. Illegitimate recombination might be carried out by a general mechanism in organisms ranging from prokaryotes to higher eukaryotes.

Animals↗

PubMed1988

Studies on the inhibitory effect of tetrandrine on silica-induced fibrogenesis in vitro.

Two cell culture systems composed of rat peritoneal macrophages and 2BS fibroblasts were used to study the inhibitory action of tetrandrine on collagen and glycosaminoglycan synthesis. The activation of silica-treated macrophage supernatants stimulated collagen synthesis by 2BS fibroblast cells. Tetrandrine had an inhibitory effect on the biosynthesis of collagen and glycosaminoglycans. The increase in the content of hydroxyproline, representing collagen, was verified by comparing it with that of DNA.

Alkaloids↗

PubMed1987

Growth-related elevations of DNA topoisomerase II levels found in Dunning R3327 rat prostatic adenocarcinomas.

The relationship between DNA topoisomerase II expression and mammalian cell proliferation has been evaluated by determining enzyme levels in normal and neoplastic rat prostate tissues. By activity assay and by immunoblot analysis using anti-topoisomerase II antiserum, topoisomerase II levels were found to be elevated in both the Dunning R3327-H and the Dunning R3327-G rat prostatic adenocarcinomas over levels assayed in the normal rat dorsal prostate. Immunohistochemical studies using the antitopoisomerase II antiserum revealed that a greater fraction of nuclei contained detectable levels of topoisomerase II in tissue sections prepared from each of the Dunning tumors than in rat dorsal prostate tissue sections. The Dunning R3327-H and R3327-G tumors grow at different rates in vivo (J. T. Isaacs and D. S. Coffey, Clin. Oncol., 2: 479-498, 1983). When measured topoisomerase II levels were compared to known growth parameters for each of the tissues studied, topoisomerase II expression was found to be correlated with tissue growth rate.

Adenocarcinoma↗

PubMed1987

Supercoiling of the DNA template during transcription.

Transcription of a right-handed double-helical DNA requires a relative rotation of the RNA polymerase and its nascent RNA around the DNA. We describe conditions under which the resistance to the rotational motion of the transcription ensemble around the DNA can be large. In such cases, the advancing polymerase generates positive supercoils in the DNA template ahead of it and negative supercoils behind it. Mutual annihilation of the positively and negatively supercoiled regions may be prevented by anchoring points on the DNA to a large structure, or, in the case of an unanchored plasmid, by the presence of two oppositely oriented transcription units. In prokaryotes, DNA topoisomerase I preferentially removes negative supercoils and DNA gyrase (topoisomerase II) removes positive ones. Our model thus provides an explanation for the experimentally observed high degree of negative or positive supercoiling of intracellular pBR322 DNA when DNA topoisomerase I or gyrase is respectively inhibited. We discuss the implications of our model in terms of supercoiling regulation, DNA conformational transitions, and gene regulation in both prokaryotes and eukaryotes.

DNA Topoisomerases, Type I↗

PubMed1987

Roles of DNA topoisomerases in simian virus 40 DNA replication in vitro.

We examined the roles of DNA topoisomerases in the replication of simian virus 40 (SV40) DNA in a cell-free system composed of an extract from HeLa cells supplemented with purified SV40 tumor antigen. When the activities of both topoisomerase I (EC 5.99.1.2) and topoisomerase II (EC 5.99.1.3) in the extract were blocked with specific inhibitors or antibodies, DNA synthesis was decreased by a factor of 15-20. Addition of purified HeLa DNA topoisomerase II to extracts immunologically depleted of both topoisomerases completely restored replication, and the replication products consisted largely of monomeric daughter molecules. Addition of purified HeLa DNA topoisomerase I to depleted extracts restored DNA synthesis, but the primary products were multiply intertwined, catenated daughter molecules. We conclude that DNA topoisomerases have at least two roles in the replication of SV40 DNA. Either topoisomerase I or topoisomerase II is sufficient to provide the unlinking activity necessary for fork propagation during SV40 DNA replication. However, topoisomerase II is uniquely required for the segregation of newly synthesized daughter molecules.

DNA Replication↗

PubMed1987

Integration of the DNA of filamentous bacteriophage Cflt into the chromosomal DNA of its host.

It was demonstrated for the first time that filamentous bacteriophage Cflt, which contains single-stranded DNA, can incorporate its genome into that of its host. Evidence in support of the incorporation was obtained from a Southern blot hybridization analysis of DNA isolated from Cflt-lysogenized cells. DNAs from different Cflt-lysogenized cells were purified, and the integration patterns were compared. Because all integration patterns were identical and only one fragment in Cflt replicative-form DNA was missing, it appears that the integration was site specific. Only one complement of viral DNA was integrated per host chromosome. To determine the attachment site on the viral DNA, the physical map of EcoRI, XhoI, SstII, and BglII on Cflt DNA was constructed. Based on this physical map and a Southern blot hybridization analysis of lysogen DNA with these restriction endonucleases, we demonstrated that DNA sequences from all regions of the Cflt genome were represented in the integrated viral sequences. The attachment site on the viral genome was located at 69.2 to 73.8 min on the Cflt DNA.

Bacteriophages↗

PubMed1987

Regulation of DNA topoisomerases during cellular differentiation.

DNA topoisomerase II has been shown to be a nuclear marker for cell proliferation and a therapeutic target in cancer chemotherapy. In order to study the regulation of DNA topoisomerases during cellular differentiation, MELC were induced to differentiate by treatment with 5 mM HMBA. At day five, approximately 95% of MELC had reproducibly undergone differentiation. The level of topoisomerase II, as measured by immunoblotting with anti-topoisomerase II antisera, showed a parallel decrease to approximately 5-10% of the control level by day five. Activity measurements showed a similar decrease during the time course of MELC differentiation. Immunofluorescence studies at day five showed that only about 5% of the MELC had strong nuclear immunofluorescence. These results indicate that the level and activity of DNA topoisomerase II are significantly lower in differentiated versus undifferentiated cells. We also observed that the level and activity of topoisomerase II dropped twofold as cells grew to high cell densities in the absence of HMBA. In contrast, the topoisomerase I levels in MELC remained relatively constant throughout growth and differentiation.

Animals↗

PubMed1987

Involvement of intracellular ATP in cytotoxicity of topoisomerase II-targetting antitumor drugs.

The effect of the ATP pool on the cytotoxic action of teniposide (VM-26) has been studied in mouse leukemia cells (L1210). L1210 cells in tissue culture were treated with VM-26 (10 microM) in the presence of DNP, an uncoupler of oxidative phosphorylation. The simultaneous treatment of DNP (1 mM) increased cell survival 100-200 fold. Pre- or post-treatment with DNP had little effect on cell survival. Other uncouplers and inhibitors of ATP synthesis had effects similar to DNP. The interference of DNP with the cytotoxic action of VM-26 was also seen with another topoisomerase II-targetting drug, m-AMSA, but not with the topoisomerase I-targetting drug camptothecin. Studies using either purified topoisomerase II or cultured mammalian cells had shown that DNP had little effect on the amount of cleavable complexes induced by VM-26. We propose that an ATP requiring process(es) which occurs subsequent to the formation of the cleavable complexes is involved in the cytotoxic action of topoisomerase II-targetting drugs.

Adenosine Triphosphate↗

PubMed1987

DNA topoisomerase II as a potential factor in drug resistance of human malignancies.

The stabilization of the cleavable complex between DNA topoisomerase II and DNA by adriamycin (ADR), as well as by other topoisomerase II-targeted drugs, is an essential step in a process associated with drug cytotoxicity. Unlike many other cell types, ADR does not produce DNA cleavage in the lymphocytes of chronic lymphocytic leukemia (CLL). The CLL lymphocytes have been identified as quiescent cells with an extremely low level of topoisomerase II. The low level of this enzyme could constitute a basis for a new mechanism of drug resistance operating not only in CLL, but perhaps in any slow growing cancer with a large population of quiescent cells. Other factors contributing to drug resistance could include changes in enzyme regulation or processing of the cleavable complex, or the presence of a "mutant" enzyme which renders cancer cells unresponsive to topoisomerase II-targeted drugs. Suggested strategies in drug development, aimed at the topoisomerase II-related drug resistance, could include 1) the selection of topoisomerase I as an alternative target for cancer chemotherapy, 2) the development of ADR analogs which, unlike ADR, stabilize the topoisomerase II-DNA complex with high efficiency, and 3) the search for agents enhancing the SOS-like repair response, presumably triggered by DNA topoisomerase-targeted drugs.

Animals↗

PubMed1987

Metabolic activation of N-acylanthracyclines precedes their interaction with DNA topoisomerase II.

The N-acylanthracyclines AD32 (N-trifluoroacetyladriamycin-14-valerate) and AD143 (N-trifluoroacetyladriamycin-14-O-hemiadipate) are analogs of Adriamycin (ADR) undergoing clinical or advanced pre-clinical screening. Their principal metabolites, following the cleavage of the 14-acyl side-chain, are N-trifluoroacetyladriamycin (AD41) and its reduced form N-trifluoroacetyladriamycinol (AD92). Both these compounds are biologically active and detectable in treated patients, laboratory animals, and in tissue culture cells. Unlike ADR, AD32, as well as AD143 and metabolites, show no detectable binding to double-strand DNA. Their effects on DNA have been previously investigated in vivo and in vitro using the alkaline filter-elution assay. It has been shown that all of the compounds cause approximately equivalent amounts of protein-associated DNA breaks (PAB) and DNA-protein crosslinks in a mouse lymphoma and in tissue-culture leukemia cells. In order to establish whether the induction of PAB by the drugs requires DNA topoisomerase II mediation, cleavage mapping analysis was done with tested compounds using purified human topoisomerase II. DNA fragmentation was significantly enhanced in the presence of the enzyme and either AD41 or AD92. In contrast, no fragmentation enhancement was observed in the presence of the parental drugs AD32 or AD143. The results strongly suggest that metabolic activation of N-acylanthracyclines by nonspecific esterases is a prerequisite for their interaction with DNA topoisomerase II and for stabilization of the cleavable complex.

Animals↗

PubMed1986

Induction of sister chromatid exchanges by inhibitors of topoisomerases.

To investigate the role of topoisomerases in the production of sister chromatid exchanges, the effects of inhibitors of type I and II topoisomerases on baseline and mutagen-induced sister chromatid exchanges were compared. V79 cells were treated with VM-26 and m-AMSA, known inhibitors of type II topoisomerase, or with camptothecin, the only known inhibitor of type I topoisomerase. We observed that inhibitors of both type I and II topoisomerases induced high levels of sister chromatid exchanges at 10(-6) M, and that the dose-response curves of these drugs were very similar. A clear heterogeneity in the distribution patterns of exchanges induced by inhibitors of topoisomerases was observed. We believe that this heterogeneity in response to these compounds is due to variation in sensitivity within the cell cycle. We also studied interactions of these agents with mitomycin-C and with PUVA (8-methoxypsoralen + UVA), both cross-linking agents and potent sister chromatid exchange inducers, and with x-rays, an agent that induces high levels of DNA strand breaks. No significant change in exchange levels was observed in interactions between topoisomerase inhibition and the levels induced by the agents studied. We conclude that double-strand break prevalence, known to be increased through inhibition of type II topoisomerase, is not the primary mechanism for induction of sister chromatid exchanges. We further conclude that acute inhibition of type I and type II topoisomerases does not influence substantially the induction of exchanges by other agents.

Amsacrine↗

PubMed1986

In vivo localization of DNA topoisomerase II cleavage sites on Drosophila heat shock chromatin.

Similar to its inhibitory effect on mammalian DNA topoisomerase II, the cytotoxic drug VM26 (teniposide) also interferes with the breakage-reunion reaction of Drosophila melanogaster DNA topoisomerase II. VM26 induces topoisomerase II-mediated DNA breakage in vitro and in cultured D. melanogaster cells presumably by stabilizing an enzyme-DNA cleavable complex. The drug-induced DNA breaks on D. melanogaster hsp70 genes were mapped in cultured cells using the indirect end-labeling procedure. Multiple and specific cleavage sites occurred at both the 3' and 5' ends of the hsp70 genes. A number of these cellular topoisomerase II cleavage sites mapped close to the DNase I-hypersensitive regions of the hsp70 genes. The intensities of several topoisomerase II cleavage sites changed significantly on heat shock induction. Treatment of cultured D. melanogaster cells with VM26 at 25 degrees C resulted in the stimulation of transcription of the hsp70 genes. These results suggest that inhibition of DNA topoisomerase II may lead to heat shock transcription.

Animals↗

PubMed1986

DNA damage by antitumor acridines mediated by mammalian DNA topoisomerase II.

Antitumor drugs from many chemical classes have been shown to induce protein-linked DNA breaks in cultured mammalian cells and in vitro in the presence of purified mammalian DNA topoisomerase II. The possibility that mammalian DNA topoisomerase II is an intracellular target which mediates drug-induced DNA breaks is supported by the following studies using 4'-(9-acridinylamino)methane-sulfon-m-anisidide (m-AMSA): (a) a single m-AMSA-dependent DNA cleavage activity copurified with calf thymus DNA topoisomerase II activity at all chromatographic steps of the enzyme purification; (b) m-AMSA-induced DNA cleavage by this purified activity resulted in the covalent attachment of protein to the 5'-ends of the DNA via a tyrosyl phosphate bond. This covalently linked protein has the same reduced molecular weight as purified calf thymus DNA topoisomerase II. The possibility that topoisomerase II-mediated DNA breaks may be responsible for cytotoxicity has also been investigated using a number of m-AMSA-related acridines. The level of topoisomerase II-mediated DNA breaks in vitro strongly correlates with the level of protein-linked DNA breaks in cultured cells and drug-induced cytotoxicity. These results suggest that mammalian DNA topoisomerase II may be a cytotoxic target of antitumor acridines.

Acridines↗

PubMed1985

Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I.

Camptothecin, a cytotoxic drug, is a strong inhibitor of nucleic acid synthesis in mammalian cells and a potent inducer of strand breaks in chromosomal DNA. Neither the equilibrium dialysis nor the unwinding measurement indicates any interaction between camptothecin and purified DNA. However, camptothecin induces extensive single strand DNA breaks in reactions containing purified mammalian DNA topoisomerase I. DNA breakage in vitro is immediate and reversible. Analyses of camptothecin-induced DNA breaks show that topoisomerase I is covalently linked to the 3' end of the broken DNA. In addition, camptothecin inhibits the catalytic activity of mammalian DNA topoisomerase I. We propose that camptothecin blocks the rejoining step of the breakage-reunion reaction of mammalian DNA topoisomerase I. This blockage results in the accumulation of a cleavable complex which resembles the transient intermediate proposed for eukaryotic DNA topoisomerase I. The inhibition of nucleic acid synthesis and the induction of DNA strand breaks observed in vivo may be related to the formation of this drug-induced cleavable complex.

Animals↗