Recent molecular studies have expanded the biological contexts in which topoisomerase II (TOP2) has crucial functions, including DNA replication, transcription and chromosome segregation. Although the biological functions of TOP2 are important for ensuring genomic integrity, the ability to interfere with TOP2 and generate enzyme-mediated DNA damage is an effective strategy for cancer chemotherapy. The molecular tools that have allowed an understanding of the biological functions of TOP2 are also being applied to understanding the details of drug action. These studies promise refined targeting of TOP2 as an effective anticancer strategy.

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Targeting DNA topoisomerase II in cancer chemotherapy

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.

https://cancerres.aacrjournals.org/content/canres/49/18/5077.full.pdf

Arrest of Replication Forks by Drug-stabilized Topoisomerase I-DNA Cleavable Complexes as a Mechanism of Cell Killing by Camptothecin

Manual for the Identification of Medical Bacteria.

Phospholipids play multiple roles in cells by establishing the permeability barrier for cells and cell organelles, by providing the matrix for the assembly and function of a wide variety of catalytic processes, by acting as donors in the synthesis of macromolecules, and by actively influencing the functional properties of membrane-associated processes. The function, at the molecular level, of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin in specific cellular processes is reviewed, with a focus on the results of combined molecular genetic and biochemical studies in Escherichia coli. These results are compared with primarily biochemical data supporting similar functions for these phospholipids in eukaryotic organisms. The wide range of processes in which specific involvement of phospholipids has been documented explains the need for diversity in phospholipid structure and why there are so many membrane lipids.

MOLECULAR BASIS FOR MEMBRANE PHOSPHOLIPID DIVERSITY: Why Are There So Many Lipids?

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.

https://cancerres.aacrjournals.org/content/canres/48/7/1722.full.pdf

Identification of Mammalian DNA Topoisomerase I as an Intracellular Target of the Anticancer Drug Camptothecin

DNA topoisomerase I was purified to near homogeneity from a clonal line of human lymphoblastic leukemia cells, RPMI 8402, that is resistant to camptothecin, a cytotoxic alkaloid from Camptotheca acuminata, and compared with that of the parent wild-type cells. As assayed by relaxation of the supercoiled plasmid DNA and by formation of enzyme-linked DNA breaks, the purified enzyme from the resistant cells was shown to be greater than 125-fold as resistant to camptothecin as the wild-type enzyme, comparable to a cellular resistance index of about 300. Therefore, the cellular resistance appears to be due to the resistance of the enzyme. The amount of the immunoreactive enzyme protein in whole extract appeared to be reduced to less than half that of the wild-type enzyme. These results establish that DNA topoisomerase I is the cellular target of camptothecin and that DNA topoisomerase I is essential for the survival of mammalian cells.

https://www.pnas.org/content/pnas/84/16/5565.full.pdf

Characterization of a mammalian mutant with a camptothecin-resistant DNA topoisomerase I.

Several recently developed derivatives of bis(2,6-dioxopiperazine) have been shown to be new antitumor agents and are currently under clinical trials. We found that the mother compound of the bis(2,6-dioxopiperazine)s, ICRF-154, and its derivatives, ICRF-159, ICRF-193, and MST-16, are all inhibitors of mammalian type II DNA topoisomerase. By decatenation assay using kinetoplast DNA from Crithidia fasciculata, inhibition of purified calf thymus topoisomerase II by these compounds was investigated. Potency of inhibition was in the following order: ICRF-193 greater than ICRF-154 = ICRF-159 greater than MST-16. The doses giving 50% inhibition were 2, 13, 30 and 300 microM, respectively, for these compounds. ICRF-193, the most potent inhibitor, however, did not inhibit topoisomerase I at concentrations up to 300 microM. Addition of excess enzyme, but not of the substrate DNA, overcame the inhibition by ICRF-193. The drug did not stimulate the formation of cleavable complex between DNA and the enzyme. Furthermore, ICRF-193 even inhibited the formation of enzyme-mediated DNA cleavage induced by etoposide or 4'-[9-acridinylamino)methanesulfon-m-anisidide. These observations, together with the finding that ICRF-193 did not intercalate into DNA, suggest that ICRF-154 and related compounds are specific inhibitors of topoisomerase II with different modes of action: i.e., they interfere with some step(s) before the formation of the intermediate cleavable complex in the catalytic cycle. This is a property quite distinct from previously known cleavable complex-forming type topoisomerase II-targeting antitumor agents such as acridines, anthracyclines, and epipodophyllotoxins, but rather, mechanistically similar to the recently reported group of inhibitors that includes merbarone, aclarubicin, and fostriecin.

https://cancerres.aacrjournals.org/content/canres/51/18/4903.full.pdf

Inhibition of topoisomerase II by antitumor agents bis(2,6-dioxopiperazine) derivatives.

In the accompanying paper (K. Tanabe, Y. Ikegami, R. Ishida, and T. Andoh, Cancer Res., 51: 4903–4908, 1991), we showed that ICRF-154 and -193, dioxopiperazine derivatives, inhibited the activity of purified topoisomerase II, without formation of a cleavable DNA-protein complex. In order to see whether ICRF-154 and ICRF-193 affect cellular topoisomerase II in situ or not, we examined the effect of these drugs on etoposide (VP-16)-induced, topoisomerase II-mediated DNA breaks in RPMI 8402 cells by alkaline sedimentation analysis. When RPMI 8402 cells were exposed to VP-16 in the presence of ICRF-154 or ICRF-193 for 1 h, VP-16-induced DNA strand breaks were greatly inhibited by both ICRF compounds. In parallel with this observation, VP-16-induced growth inhibition was also reversed by ICRF-193. Exposure of cells to ICRF-154 resulted in a progressive accumulation of cells with 4C DNA content. Although mitotic index did not significantly increase, mitotic abnormalities were seen in cells exposed to ICRF-193 or ICRF-154: all mitotic cells exhibited early mitotic figures with fewer condensed and entangled chromosomes. The most sensitive phase of the cell cycle to ICRF-154 was the G2-M. ICRF-154 did not affect the spindle formation. However, abnormally oriented spindles were observed in drug-treated cells in parallel with the appearance of multinucleated cells. The results suggest that ICRF-154 and -193 inhibit topoisomerase II activity in RPMI 8402 cells, and this effect resulted in the appearance of cells in G2 and early M phase with fewer condensed and entangled chromosomes and of cells with multilobed nuclei.

https://cancerres.aacrjournals.org/content/canres/51/18/4909.full.pdf

Inhibition of intracellular topoisomerase II by antitumor bis(2,6-dioxopiperazine) derivatives: mode of cell growth inhibition distinct from that of cleavable complex-forming type inhibitors.

Camptothecin (CPT), a plant alkaloid with antitumor activity, is a specific inhibitor of eukaryotic DNA topoisomerase I. We have previously isolated and characterized a CPT-resistant topoisomerase I isolated from a CPT-resistant human leukemia cell line, CPT-K5. cDNA clones of topoisomerase I were isolated from the CPT-resistant and the parental CPT-sensitive cell lines, respectively. Sequencing of the clones identified two mutations in the cDNA isolated from the resistant cells, which cause amino acid changes from aspartic acid to glycine at residues 533 and 583 of the parental topoisomerase I. When the CPT-K5 topoisomerase I was expressed in E. coli as a fusion protein with Staphylococcal Protein A fragment, the activity was resistant to CPT at a dose level up to 125 microM, whereas the parental fusion protein was sensitive to CPT as low as 1 microM. The resistance index (greater than 125) of the CPT-K5 fusion topoisomerase I is similar to that of the native CPT-K5 topoisomerase I. These results indicate that either or both of the two amino acid changes identified in the mutant enzyme is responsible for the resistance to CPT.

Toshiwo Andoh

Papers

Characterization of a mammalian mutant with a camptothecin-resistant DNA topoisomerase I.

Inhibition of topoisomerase II by antitumor agents bis(2,6-dioxopiperazine) derivatives.

Inhibition of intracellular topoisomerase II by antitumor bis(2,6-dioxopiperazine) derivatives: mode of cell growth inhibition distinct from that of cleavable complex-forming type inhibitors.

Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites

Characterization of a camptothecin-resistant human DNA topoisomerase I.