Ryoji Ishida

Catalytic inhibitors of DNA topoisomerase II

Catalytic inhibitors of mammalian DNA topoisomerase II have been found recently in natural and synthetic compounds. These compounds target the enzyme within the cell and inhibit various genetic processes involving the enzyme, such as DNA replication and chromosome dynamics, and thus proved to be good probes for the functional analyses of the enzyme in a variety of eukaryotes from yeast to mammals. Catalytic inhibitors were shown to be antagonists against topoisomerase II poisons. Thus bis(2,6-dioxopiperazines) have a potential to overcome cardiac toxicity caused by potent antitumor anthracycline antibiotics such as doxorubicin and daunorubicin. ICRF-187, a (+)-enantiomer of racemic ICRF-159, has been used in clinics in European countries as cardioprotector. Furthermore, bis(2,6-dioxopiperazines) enhance the efficacy of topoisomerase II poisons by reducing their side effects in preclinical and clinical settings. Bis(2,6-dioxopiperazines) per se among others have antitumor activity, and one of their derivatives, MST-16 or Sobuzoxane, bis(N1-isobutyloxycarbonyloxymethyl-2, 6-dioxopiperazine), has been developed in Japan as an anticancer drug used for malignant lymphomas and adult T-cell leukemia in clinics.

Decreased thymosin β4 in apoptosis induced by a variety of antitumor drugs

As many antitumor drugs can kill tumors through the induction of apoptosis, the effect of these drugs presumably would be enhanced if they were used in combination with other drugs that interact with apoptotic processes. To clarify the biological events involved in the induction of apoptosis, we examined changes in the proteins associated with induction of apoptosis by antitumor drugs. When Molt-4 cells were exposed to the antitumor drugs etoposide, meso-2,3-bis(3,5-dioxopiperazine-1-yl)butane (ICRF-193), and neocarzinostatin, they exhibited apoptotic cell death as determined by flow cytometry using fluorescein isothiocyanate (FITC)-labeled annexin V staining of phosphatidylserine on membranes and detection of hypodiploid cells. Following the induction of apoptosis, a low molecular weight protein that was identified to be thymosin beta4 by HPLC analysis was commonly decreased, and the morphology of actin filaments changed into clump formations. These results suggest that decreased thymosin beta4 is involved in the induction of apoptosis by antitumor drugs.

Threonine 1342 in human topoisomerase IIalpha is phosphorylated throughout the cell cycle.

To investigate the relationship between the modulation of topoisomerase II activity and its phosphorylation state during the cell cycle, a monoclonal antibody against C-terminal peptide (residues 1335-1350) of topoisomerase IIα containing a consensus sequence of casein kinase II, TDDE and its phosphorylated threonine were prepared. In an enzyme-linked immunosorbent assay, the antibody, named PT1342, recognized the immunogenic phosphopeptide but not the non-phosphorylated form of the peptide. The PT1342 antibody reacted only with a 170-kDa protein from HeLa cells and recognized anti-topoisomerase IIα immunoprecipitants. Furthermore, the antibody did not react with the human topoisomerase IIα mutated at codon 1342 from threonine to alanine, showing that PT1342 was directed against the phosphorylated threonine 1342. To examine the level of phosphorylation of threonine 1342 of topoisomerase IIα through the cell cycle, HeLa cells were stained simultaneously for phosphorylated topoisomerase IIα and DNA and analyzed by flow cytometry. Cells in the G2-M phase contained about double the PT1341-reacted topoisomerase IIα than did cells in G1 or S phases. The antibody stained the nuclei in interphase and mitotic chromosomes and its periphery, as seen with anti-topoisomerase IIα antibody. Thus, threonine 1342 in topoisomerase IIα is phosphorylated throughout the cell cycle.

Antitumor activity of 2-amino-4,4alpha-dihydro-4alpha, 7-dimethyl-3H-phenoxazine-3-one against Meth A tumor transplanted into BALB/c mice

We examined the in vivo effect of 2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3- one (Phx) on Meth A carcinoma cells transplanted into BALB/c mice, in terms of both antitumor activity and side effects. Phx, which was synthesized by the reaction of 2-amino-5-methylphenol with bovine hemolysates, was administered i.p. at doses of 1 and 5 mg/kg to BALB/c mice transplanted with Meth A tumor cells. Phx exerted a strong antitumor activity to Meth A tumor growing in the mice as 5-fluorouracil (5-FU) did. The antitumor activity of Phx at the dose of 5 mg/kg was comparable to that of 5-FU at the dose of 7.8 mg/kg. In contrast, unlike 5-FU, Phx did not cause leukopenia while showing a strong antitumor activity. The compound also produced little changes in body weight and no wasting of mice developed. These results show that Phx has strong anti-tumor activity, but exerts lower side effects and suggest that Phx is available for therapeutic purposes in the future.

ICRF-193, a catalytic inhibitor of DNA topoisomerase II, delays the cell cycle progression from metaphase, but not from anaphase to the G1 phase in mammalian cells

We have shown previously that ICRF‐193, a catalytic inhibitor of DNA topoisomerase II (topo II), delays cell cycle progression in HeLa S3 cells. We report here that the delay of the transition in M phase is observed when HeLa S3 cells were treated with ICRF‐193 during metaphase, but not thereafter. ICRF‐193 also delayed the degradation of cyclin B in the transition from M to G1 phase, while in Chinese hamster ovary (CHO) cells the drug did not delay the progression in M phase. Since HeLa S3 and CHO cells are ‘stringent’ and ‘relaxed’ in mitotic control, respectively, it is suggested that under topo II inhibition, the M phase checkpoint operates through an inability for chromosome segregation.

Antitumor activity of 2-amino-4,4 alpha-dihydro-4 alpha, 7-dimethyl-3H-phenoxazine-3-one, a novel phenoxazine derivative produced by the reaction of 2-amino-5-methylphenol with bovine hemolysate.

2-Amino-4,4 alpha-dihydro-4 alpha,7-dimethyl-3H-phenoxazine-3-one (Phx) was synthesized by the reaction of 2-amino-5-methyl-phenol with bovine hemolysates. Since Phx is a phenoxazine derivative like actinomycin D, which exerts a strong anti-tumor effect by intercalating DNA, we examined the effects of Phx on cell proliferation and cell cycle progression in human epidermoid carcinoma cells (KB cells). Phx inhibited the proliferation of Kb cells in a dose-dependent manner. When KB cells were incubated for 9 h with medium containing 50 microM Phx, a transient accumulation of cells in S and G2/M phase was observed and at 24 h many of cells had lower DNA content. Although Phx had antitumor activity, the drug did not intercalate DNA, showing a different mode of action from actinomycin D.

An improved method for the rapid preparation of 2-amino-4,4a-dihydro-4a,7-dimethyl-3H-phenoxazine-3-one, a novel antitumor agent.

A simple and rapid preparation method for a novel antitumor agent, 2-amino-4,4a-dihydro-4a,7-dimethyl-3H-phenoxazine-3-one (Phx) was described. The procedure included (1) the reaction of bovine hemolysates with 2-amino-5-methylphenol, (2) one-shot denaturation of hemoglobin and proteins by methanol, and removal of the denatured hemoglobin and proteins, (3) concentration of the reaction products, and (4) purification by a Sephadex column. These procedures yielded Phx in 34% yield.

Cross-resistance of novobiocin-resistant BHK cell line to topoisomerase II inhibitors

A novobiocin-resistant BHK cell line, designated as Novr A2, was found to exhibit cross-resistance to other topoisomerase II inhibitors such as 4′-dimethylepipodophyllotoxin-4-(4,6-O-ethylidine-β-d-glucopyranoside) (VP-16), adriamycin, and 4′-(9-acridinyl-ami-no)methanesulfon-m-anisidide (m-AMSA), and also to different types of drugs such as vinblastine and arabinocytidine. Nalidixic acid-resistant cells (A2Nalr) of the NovrA2 cell line were phenotypically reverted to novobiocin sensitivity like wild-type cells and were also partially reverted to sensitivity to VP-16 and adriamycin, but not to vinblastine and arabinocytidine. When VP-16 was added to cell culture, the drug-induced DNA strand breaks were much fewer in NovrA2 cells than in BHK cells. This reduced level of strand breaks in NovrA2 cells was not due to reduced drug uptake, because the two cell lines accumulated similar levels of radiolabeled VP-16. VP-16 also induced fewer DNA breaks in isolated nuclei of NovrA2 cells than in those of BHK cells. There was no significant difference in the VP-16-induced DNA cleavage activities of partially purified topoisomerase II from BHK and Novr cells. These results show that the resistance of NovrA2 cells to various drugs is not acquired by a defense mechanism related to membrane permeability and suggest that the resistance of the NovrA2 cells to topoisomerase II inhibitors might be due in part to alteration in a topoisomerase II associated factor(s).

Isolation and characterization of novobiocin-resistant BHK cells

We isolated two novobiocin-resistant mutants which were stable and approximately three and four times more resistant than the parent cells to novobiocin. Both mutants (Novr A2, Novr) A41 were more sensitive than the wild-type cells to nalidixic acid, and cold sensitive for cell growth. When we isolated derivatives of Novr A2 and Novr A41 cells which are resistant to nalidixic acid, those are found to be phenotypically reverted to novobiocin sensitivity like wild-type cells, thereby suggesting the relationship between the targets for novobiocin and for nalidixic acid. But the cold sensitivity did not always revert to wild type, with accompanying resistance to nalidixic acid. The DNA and RNA syntheses of Novr mutants were more resistant to novobiocin but more sensitive to nalidixic acid, than those of wild-type cells. However, in vitro assays of wild-type and Novr cell extracts were unable to demonstrate any differences in the sensitivity of topoisomerase II activity to inhibition by novobiocin. While the targets of novobiocin and nalidixic acid show a mutual interaction in vivo and play a role in DNA replication and transcription, our results suggest that these targets are probably not topoisomerase II.

Biochemical and genetic analysis of toxic effect of HOE 15030 in mammalian cells

HOE 15030 inhibited the growth of BHK cells at concentrations that did not inhibit their nuclear DNA and RNA syntheses. When BHK cells were cultured in the presence of 30 μg/ml of HOE 15030, cells were arrested in the G1 phase after one or two cell divisions. After removal of the drug, cells progressed through the G1 to the S phase. HOE 15030 inhibited the activities of both topoisomerases I and II in vitro. To determine the target molecule of HOE 15030 in cells, we isolated a HOE 15030-resistant (HOEr) mutant of BHK cells. The HOEr cells exhibited cross-resistance to ethidium bromide, acriflavine, and rhodamine 123, and slight cross-resistance to 4′-dimethylepipodophyllotoxin-4-(4,6-O -ethylidine-β-d-glucopyranoside) (VP-16) and adriamycin, but not to chloramphenicol, oligomycin, novobiocin, colchicine, or vinblastine. The uptake and retention of rhodamine 123 by HOEr cells were lower than those by BHK cells. Mitochondrial DNA synthesis of HOEr cells was more resistant to HOE 15030 and ethidium bromide than that of wild-type cells. These results indicate that the resistance of HOEr cells to drugs is due to reduced uptake or accumulation of the drugs by mitochondria and suggest that the mitochondria are the main target of HOE 15030 in cells.