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L F Liu

Publications and source records attributed to L F Liu.

At least 127 records · Page 7Linked to original sources
PubMed1989

Topoisomerase-targeting antitumor drugs.

Much has been learned about the unusual type of DNA damage produced by the topoisomerases. The mechanism by which these lesions trigger cell death, however, remains unclear, but it appears that DNA metabolic machinery transforms reversible single-strand cleavable complexes to overt strand breaks which may be an initial event in the cytotoxic pathway. For the topoisomerase I poisons, they produce breaks at replication forks that appear to be the equivalent of a break in duplex DNA. Indicating that this may be an important cytotoxic lesion is the hypersensitivity to camptothecin of the yeast mutant rad52, which is deficient in double-strand-break-repair. The topoisomerase poisons preferentially kill proliferating cells. In the case of the topoisomerase I poison camptothecin, dramatic S-phase-specific cytotoxicity can explain its preferential action on proliferating cells. For the topoisomerase II poisons, high levels of the enzyme in proliferating cells, and very low levels in quiescent cells appear to explain the resistance of quiescent cells to the drug's cytotoxic effects. Thus, the topoisomerase poisons convert essential enzymes into intracellular, proliferating-cell toxins. The identification of both topoisomerase I and II as the specific targets of cancer chemotherapeutic drugs now provides a rational basis for the development of topoisomerase I poisons for possible clinical use. Knowledge of the molecular mechanisms of cell killing may lead to the identification of new therapies for treating cancer. The topoisomerase poisons appear to be a good tool for studying cell killing mechanisms as they produce highly specific and reversible lesions.

Animals↗
PubMed1989

DNA topoisomerase I--targeted chemotherapy of human colon cancer in xenografts.

Drug development is needed to improve chemotherapy of patients with locally advanced or metastatic colon carcinoma, who otherwise have an unfavorable prognosis. DNA topoisomerase I, a nuclear enzyme important for solving topological problems arising during DNA replication and for other cellular functions, has been identified as a principal target of a plant alkaloid 20(S)-camptothecin. Significantly increased concentrations of this enzyme, compared to that in normal colonic mucosa, were found in advanced stages of human colon adenocarcinoma and in xenografts of colon cancer carried by immunodeficient mice. Several synthetic analogs of camptothecin, selected by tests with the purified enzyme and tissue-culture screens, were evaluated in the xenograft model. Unlike other anticancer drugs tested, 20(RS)-9-amino-camptothecin (9-AC) induced disease-free remissions. The overall drug toxicity was low and allowed for repeated courses of treatment.

Adenocarcinoma↗
PubMed1989

Poly(ADP-ribose)-mediated post-translational modification of chromatin-associated human topoisomerase I. Inhibitory effects on catalytic activity.

We have investigated the association of human topoisomerase I with poly(ADP-ribosylated) domains of chromatin and the effects of this modification on the enzyme activity. In vitro poly(ADP-ribosylation) assays demonstrated that this enzyme was one of the major acceptors for this chromatin-dependent post-translational modification. Western blotting procedures using antibody to topoisomerase I indicated that under extensive poly(ADP-ribosylation) conditions, where a majority of poly(ADP-ribose) acceptor molecules form aggregates, the major population of the topoisomerase I associated with chromatin was apparently non-aggregated. The catalytic activity of the topoisomerase I associated with the poly(ADP-ribosylated) chromatin was 3-5-fold inhibited. Additionally, antibody to poly(ADP-ribose) was used to immunofractionate selectively the modified domains of chromatin. Our data suggests the presence of topoisomerase I, both adjacent and distal to the poly(ADP-ribosylated) sites of chromatin. Unmodified and a significant portion of the modified species of enzyme migrated as approximately 100-kDa proteins. However, the modified form of topoisomerase was noted to be catalytically less active as compared to the enzyme bound to the non-poly(ADP-ribosylated) nucleosomes. These results provide evidence, at the cellular level, for the poly(ADP-ribosylation)-mediated regulation of human topoisomerase I and suggest a functional significance for poly(ADP-ribosylation) in topoisomerase-related processes (replication, transcription, and recombination) in eukaryotes.

Blotting, Western↗
PubMed1989

DNA topoisomerase II-mediated interaction of doxorubicin and daunorubicin congeners with DNA.

Three groups of doxorubicin and daunorubicin analogues, differing by their substituents on the chromophore and sugar moieties, were used in this study. The 3'-N-unsubstituted (Group 1), 3'-N-acyl (Group 2), and 3'-N-alkyl (Group 3) analogues were tested for: (a) in vivo antitumor activity and in vitro cytotoxicity; (b) cellular or tissue uptake and metabolic conversion; (c) strength of DNA intercalation; and (d) interaction with DNA topoisomerase II (topo-II). Compounds of Group 1 were cytotoxic, were strongly intercalative, and, except for those with C-14 side chain substitution, induced the formation of topo-II-DNA cleavable complexes. As shown previously, esterolysis of C-14-acyl substituents was required to yield a metabolite which can interact with topo-II in the purified system. The C-14-substituted compounds of Group 2 and their C-14-unsubstituted metabolites were cytotoxic. These drugs were weak intercalators, and the C-14-unsubstituted cogeners induced cleavable complex formation in the purified system, but with reduced potency relative to doxorubicin. The type of the 3'-N-position substituent determined whether Group 3 analogues were cytotoxic and strong intercalators, or less active and nonintercalating. Although C-14-unsubstituted intercalators of Group 3 did not form cleavable complexes in the purified system, they were cytotoxic. The study shows that DNA intercalation is required but not sufficient for the activity by topo-II-targeted anthracyclines. In addition to the planar chromophore which is involved in intercalation, two other domains of the anthracycline molecule are important for the interaction with topo-II: (a) substitution of the C-14 position totally inhibits drug activity in the purified system, but enhances cytotoxicity by aiding drug uptake and presumably acting on other cellular targets; and (b) substitutions on the 3'-N position of the sugar ring can, depending on the nature of the substituent, inhibit intercalation and/or topo-II-targeting activity. These findings may provide guidance for the synthesis and development of new active analogues.

Animals↗
PubMed1989

Arrest of replication forks by drug-stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin.

Camptothecin, which induces an unusual type of DNA damage by trapping cellular topoisomerase I on chromosomal DNA in the form of drug-enzyme-DNA cleavable complexes, inhibits DNA synthesis and specifically kills S-phase cells. Cotreatment of L1210 cells with aphidicolin, which is an inhibitor of replicative DNA polymerases, completely abolished camptothecin cytotoxicity, suggesting the involvement of DNA replication in camptothecin cytotoxicity. In order to study the role of DNA replication in drug action, a cell-free SV40 DNA replication system was used in the present study. Camptothecin inhibited SV40 DNA replication in this cell-free system only in the presence of topoisomerase I. Addition of excess purified calf thymus DNA topoisomerase I to this extract system in the presence of camptothecin resulted in severe inhibition of SV40 DNA replication and the accumulation of linearized replication products, which contained covalently bound DNA topoisomerase I. We propose that the collision between moving replication forks and camptothecin-stabilized topoisomerase I-DNA cleavable complexes results in fork arrest and possibly fork breakage, which are lethal to proliferating cells.

Animals↗
PubMed1989

Expression of Yb1 glutathione S-transferase using a baculovirus expression system.

A full-length cDNA clone was isolated for rat liver Yb1 glutathione S-transferase (EC 2.5.1.18). The coding sequence of Yb1 cDNA was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells. The enzymatically active recombinant Yb1 glutathione S-transferase protein has a native molecular weight of 42,000 daltons (by molecular sieve chromatography), a subunit molecular weight of 26,500 daltons (by SDS-polyacrylamide gel electrophoresis), a pI of 8.4 and an extinction coefficient E1%280 of 5.6 +/- 0.4.

Animals↗
PubMed1989

DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues.

20(S)-Camptothecin, the 20(S)-camptothecin sodium salt, and 12 analogues with substituents on the A ring differ widely in their effectiveness in the treatment of murine L1210 lymphoblastic leukemia in vivo. The drugs were screened in the following systems: System 1, the cleavage of DNA in the presence of purified topoisomerase I; System 2, drug-induced trapping of topoisomerase I in a covalent complex with DNA; and System 3, the induction of protein-associated DNA breaks in drug-treated L1210 leukemia cells. 9-Amino-20(S), 10-amino-20(RS), and 10,11-methylenedioxy-20(RS), drugs effective against murine L1210 leukemia in vivo, stabilize topoisomerase I-DNA cleavable complexes in a purified system and in cultured L1210 cells. Other analogues, inactive against L1210 leukemia in vivo, were totally ineffective in topoisomerase I-directed screens. The rest of the analogues were intermediate in terms of their antitumor and topoisomerase I-directed activities. The study shows that the drug-induced accumulation of enzyme-DNA cleavable complexes is directly proportional to drug cytotoxicity and antitumor activity.

Animals↗
PubMed1989

Evidence for the reversibility of cellular DNA lesion induced by mammalian topoisomerase II poisons.

Like many intercalative antitumor drugs, the nonintercalative antitumor drug epipodophyllotoxin VM-26 (teniposide) induces topoisomerase II-linked DNA breaks as revealed by cell lysis with a strong protein denaturant such as sodium dodecyl sulfate or alkali. We show that the majority of topoisomerase II-linked DNA breaks reflect the formation of reversible topoisomerase II-DNA cleavable complexes in drug-treated cells by demonstrating the reversibility of this unusual type of DNA damage at elevated temperatures (e.g. 65 degrees C).

Animals↗
PubMed1989

Transcription-driven supercoiling of DNA: direct biochemical evidence from in vitro studies.

The translocation of an RNA polymerase elongation complex along double helical DNA has been proposed to generate positive supercoiling waves ahead of and negative supercoiling waves behind the transcription ensemble. This twin supercoiled domain model has been tested in vitro. In the presence of prokaryotic DNA topoisomerase I, which selectively removes negative supercoils, transcription from a single promoter results in rapid and extensive positive supercoiling of the DNA template. The accumulation of positive supercoils in the DNA template requires continued movement of the elongation complex as well as sizable nascent RNA chains. These in vitro results provide direct biochemical evidence supporting the twin supercoiled domain model of transcription. Furthermore, the magnitute of DNA supercoiling (torsional) waves generated by transcription is much greater than previously expected, suggesting that transcription is one of the principal factors affecting intracellular DNA supercoiling.

DNA Topoisomerases, Type I↗
PubMed1989

Template supercoiling during ATP-dependent DNA helix tracking: studies with simian virus 40 large tumor antigen.

Incubation of topologically relaxed plasmid DNA with simian virus 40 (SV40) large tumor antigen (T antigen), ATP, and eubacterial DNA topoisomerase I resulted in the formation of highly positively supercoiled DNA. Eukaryotic DNA topoisomerase I could not substitute for eubacterial DNA topoisomerase 1 in this reaction. Furthermore, the addition of eukaryotic topoisomerase I to a preincubated reaction mixture containing both T antigen and eubacterial topoisomerase I caused rapid relaxation of the positively supercoiled DNA. These results suggest that SV40 T antigen can introduce topoisomerase-relaxable supercoils into DNA in a reaction coupled to ATP hydrolysis. We interpret the observed T antigen supercoiling reaction in terms of a recently proposed twin-supercoiled-domain model that describes the mechanics of DNA helix-tracking processes. According to this model positive and negative supercoils are generated ahead of and behind the moving SV40 T antigen, respectively. The preferential relaxation of negative supercoils by eubacterial DNA topoisomerase I explains the accumulation of positive supercoils in the DNA template. The supercoiling assay using DNA conformation-specific eubacterial DNA topoisomerase I may be of general use for the detection of ATP-dependent DNA helix-tracking proteins.

Adenosine Triphosphate↗
PubMed1989

Topoisomerase II-mediated DNA cleavage by amonafide and its structural analogs.

Treatment of SV40-infected monkey cells with amonafide (benzisoquinolinedione), an intercalative antitumor drug, resulted in rapid accumulation of linearized intracellular SV40 DNA molecules that were protein linked. Studies using purified mammalian DNA topoisomerase II have shown that amonafide and its structural analogs interfere with the breakage-rejoining reaction of the enzyme by stabilizing a reversible enzyme-DNA "cleavable complex." Denaturation of the cleavable complex with sodium dodecyl sulfate resulted in DNA cleavage and the covalent association of topoisomerase II polypeptides with the cleaved DNA. Unwinding measurements indicate that amonafide is a DNA intercalator. These results suggest that amonafide and its structural analogs (e.g., mitonafide) represent a new class of intercalative topoisomerase II-active antitumor drugs. Different from other topoisomerase II-active antitumor drugs, amonafide and mitonafide induce specific DNA cleavage at a single major site on pBR322 DNA. The strong site specificity of amonafide may allow detailed characterization of the intercalator-stabilized, topoisomerase II-DNA cleavable complex.

Adenine↗
PubMed1988

Resistance of human leukemic and normal lymphocytes to drug-induced DNA cleavage and low levels of DNA topoisomerase II.

Adriamycin, amsacrine, and etoposide produce protein-associated DNA breaks in numerous cell types. However, in vitro exposure to Adriamycin (0.1-50.0 micrograms/ml) resulted in no detectable DNA cleavage in lymphocytes from patients with B-cell chronic lymphocytic leukemia (CLL) or in either B- or T-lymphocytes from normal donors. In contrast, DNA cleavage was observed in T-cells from CLL patients. Exposure to amsacrine or etoposide caused at least 50-fold less DNA cleavage in CLL and normal lymphocytes as compared to L1210 cells. These findings cannot be accounted for by differences in drug uptake. An attempt was made to explain the relative resistance of human lymphocytes to drug-induced DNA cleavage. DNA topoisomerase II, an intracellular target of tested drugs, was assayed in CLL and normal human blood lymphocytes by immunoblotting. The enzyme was detected neither in unfractionated lymphocytes nor in the enriched B- and T-cells from 28 untreated patients with CLL (Stage 0-IV) and from seven normal donors. Exponentially growing L1210 cells had approximately 7 x 10(5) enzyme copies per cell, suggesting a 100-fold higher content than that of CLL or normal lymphocytes. There were, however, detectable levels of DNA topoisomerase II in cells obtained from patients with diffuse histiocytic, nodular poorly differentiated and nodular mixed lymphomas, in Burkitt's lymphoma, acute lymphoblastic leukemia and CLL with prolymphocytic transformation. DNA topoisomerase I, a potential target for anticancer chemotherapy, was detectable in CLL and normal lymphocytes, as well as in cells of other malignancies tested. The above results may offer an explanation for the ineffectiveness of Adriamycin in the treatment of CLL. It could be suggested that low levels of DNA topoisomerase II contribute to drug resistance operating in human malignancies with a large compartment of nonproliferating cells.

Amsacrine↗
PubMed1988

Proliferation-dependent regulation of DNA topoisomerase II in cultured human cells.

The intracellular level of DNA topoisomerase II appears to be reversibly regulated by serum concentration in cultured primary human skin fibroblasts (HSF). Upon serum starvation, the intracellular level of topoisomerase II in HSF, as monitored by immunoblotting with antitopoisomerase II antibodies, gradually decreased to a nondetectable level (less than 10(4) copies/cell) over a period of 72 h. Addition of 10% serum to the starved cells led to a gradual increase of the intracellular topoisomerase II to the original level (approximately 10(6) copies/cell) over a period of 24 h. The intracellular DNA topoisomerase II level in HSF is also sensitive to cell density; minimally a 7-fold decrease was observed when HSF were grown to saturation density in a constant serum concentration. Similarly, the intracellular levels of DNA topoisomerase II in other "nontransformed" cells such as mouse NIH 3T3 and 3T6 cells are also sensitive to both the serum concentration and the cell density. In contrast, topoisomerase II levels in transformed cells such as HeLa cells, L1210 cells, and SV40 T-antigen-transformed COS-1 cells are maintained at high levels (approximately 10(6) copies/cell) and are much less sensitive to growth conditions. The topoisomerase II level in HeLa cells synchronized by a double thymidine block remained relatively constant (less than 2-fold difference) throughout the late G1, S, G2, and M phases of the cell cycle. Our results suggest that the level of DNA topoisomerase II is primarily regulated in the G0-G1 phase of the cell cycle and is elevated to a high level (approximately 10(6) copies/cell) in proliferating cells. In contrast, the intracellular levels of DNA topoisomerase I in these cells were largely unaffected by these growth conditions either in HSF or in HeLa cells.

Cell Cycle↗
PubMed1988

Transcription generates positively and negatively supercoiled domains in the template.

We show that transcription of a DNA molecule inside a bacterium is accompanied by local and temporal supercoiling of the DNA template: as transcription proceeds, DNA in front of the transcription ensemble becomes positively supercoiled, and DNA behind the ensemble becomes negatively supercoiled. Because bacterial gyrase and topoisomerase I act differently on positively and negatively supercoiled DNA, the formation of twin supercoiled domains during transcription is manifested by a large increase or decrease in the linking number of an intracellular plasmid when bacterial DNA gyrase or topoisomerase I, respectively, is inhibited. Such changes in linking number are strongly dependent on transcription of the plasmid in cis and on the relative orientations of transcription units on the plasmid. These results indicate that the state of supercoiling of bacterial DNA is strongly modulated by transcription, and that DNA topoisomerases are normally involved in the elongation step of transcription.

DNA Topoisomerases, Type I↗
PubMed1988

Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin.

Camptothecin, a plant alkaloid with antitumor activity, has been shown to be a potent inhibitor of nucleic acid synthesis and a strong inducer of DNA strand breaks in mammalian cells. Previous studies have shown that camptothecin inhibits purified mammalian DNA topoisomerase I by trapping a reversible enzyme-DNA "cleavable complex" (Hsiang et al., J. Biol. Chem., 260: 14873-14878, 1985). Our present studies, using L1210 cells and SV40-infected monkey cells, have shown that camptothecin-induced strand breaks are protein linked. The linked protein is most likely DNA topoisomerase I as revealed by immunoblot analysis, using antibodies against purified mammalian DNA topoisomerase I. Brief heating of camptothecin-treated cells to 65 degrees C resulted in a rapid reduction of the number of protein-linked DNA breaks. Reversal of the camptothecin-induced topoisomerase I-DNA complex by heat was also observed in an in vitro system by using purified mammalian DNA topoisomerase I. Our results suggest that camptothecin interferes with DNA topoisomerase I both in cultured mammalian cells and in the purified system by trapping a reversible enzyme-DNA cleavable complex.

Animals↗
PubMed1988

Studies on the effect of tetrandrine on microtubules. I. Biochemical observation and electron microscopy.

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↗