Epolactaene, a Novel Neuritogenic Compound in Human Neuroblastoma Cells, Selectively Inhibits the Activities of Mammalian DNA Polymerases and Human DNA Topoisomerase II

Biochemical and Biophysical Research Communications 273, 784 –788 (2000) doi:10.1006/bbrc.2000.3007, available online at http://www.idealibrary.com on

Epolactaene, a Novel Neuritogenic Compound in Human Neuroblastoma Cells, Selectively Inhibits the Activities of Mammalian DNA Polymerases and Human DNA Topoisomerase II Yoshiyuki Mizushina,* Susumu Kobayashi,† Kouji Kuramochi,† Seigo Nagata,† Fumio Sugawara,* and Kengo Sakaguchi* ,1 *Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan; and †Faculty of Pharmaceutical Sciences, Science University of Tokyo, 12 Ichigaya-Funagawara-machi, Shinjuku-ku, Tokyo, 162-0826, Japan

Received May 25, 2000

We chemically synthesized epolactaene, a neuritogenic compound in human neuroblastoma cells, and investigated its biochemical action in vitro. Epolactaene and its derivatives selectively inhibited the activities of mammalian DNA polymerase ␣ and ␤ and human DNA topoisomerase II, with IC 50 values of 25, 94, and 10 ␮M, respectively. By comparison with its structural derivatives, the long alkyl side chain in epolactaene seemed to have an important role in this inhibitory effect. The compound did not influence the activities of plant or prokaryotic DNA polymerases or of other DNA metabolic enzymes such as telomerase, RNA polymerase, and deoxyribonuclease I. Epolactaene did not intercalate into DNA. These results suggested that the neuritogenic compound epolactaene influences both DNA polymerases and topoisomerase II despite the dissimilarity in both structure and properties of these two enzymes and that inhibition of these enzymes could be related to the neuritogenic effect in human neuroblastoma cells. The relationship between the neuritogenic mechanism and cell cycle regulation by epolactaene was also discussed. © 2000 Academic Press

Key Words: epolactaene; DNA polymerase ␣; DNA polymerase ␤; DNA topoisomerase II; enzyme inhibitor; neurite outgrowth.

Neurotrophic factors such as nerve growth factor are involved in survival, growth and differentiation of normal and neoplastic nerve cells (1, 2). The purpose of this study was to characterize the biochemical properties of a novel neuritogenic compound, epolactaene, in To whom correspondence should be addressed. Fax: ⫹81-471-239767. E-mail: [email protected]. 1

0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

human neuroblastoma cells. Epolactaene was screened not only as a microbial product produced by Penicillium sp. 1689-P (3), but was also independently isolated as a non-protein neurotrophic compound that induces neurite outgrowth in the human neuroblastoma cell line SH-SY5Y, which lacks significant TRK family mRNA (4, 5). Here, we report the biochemical action of epolactaene, which induces neurite outgrowth in human SH-SY5Y cells. Epolactaene significantly arrested cell cycle at G 0/G 1 phase in SH-SY5Y (6). Epolactaene may define a target molecules for the neuritogenic compounds and be useful for clarification of the relationships between neuritogenesis and cell cycle arrest. Therefore, we mainly investigated its effects on DNA metabolic enzymes including DNA polymerase and DNA topoisomerase by epolactaene, because agents that cause cell cycle arrest at a specific phase appear to influence the machinery involved in DNA synthesis. Interestingly, we found that epolactaene was not only a potent inhibitor of DNA polymerases ␣ and ␤, but also of DNA topoisomerase II. The DNA polymerases catalyze addition of deoxyribonucleotides to the 3⬘-hydroxyl terminus of primed doublestranded DNA molecules (7), and DNA topoisomerase II catalyzes the concerted breaking and rejoining of DNA strands and is involved in producing topological and conformational changes in DNA (7, 8). Therefore, there are no enzymatic similarities between these enzymes, although they are critical to many cellular processes such as DNA replication, repair and recombination, and may act in harmony with each other. MATERIALS AND METHODS Materials. Epolactaene (compound 1) and its derivatives (compound 2– 4) were chemically synthesized (Kobayashi et al., manu-

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script in preparation). Nucleotides and chemically synthesized template-primers such as poly(dA), poly(rA) and oligo(dT) 12–18 were purchased from Pharmacia (Uppsala, Sweden). [ 3H]-dTTP (43 Ci/ mmol) was purchased from New England Nuclear Corp. (Boston, MA). Supercoiled pBR322 plasmid DNA was obtained from Toyobo (Osaka, Japan). All other reagents were of analytical grade and were purchased from Wako Chemical Industries (Osaka, Japan). DNA polymerase ␣ was purified from the calf thymus by immuno-affinity column chromatography as described previously (9). Recombinant rat DNA polymerase ␤ was purified from E. coli JMp␤5 as described by Date et al. (10). DNA polymerase I (plant ␣-like polymerase) and II (plant ␤-like polymerase) from the higher plant, cauliflower inflorescence, was purified according to the methods outlined by Sakaguchi et al. (11). Purified human placenta DNA topoisomerase II (2 units/␮l) was purchased from TopoGen, Inc. (Columbus, OH). Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase and the Klenow fragment of DNA polymerase I were purchased from Worthington Biochemical Corp. (Freehold, NJ). T4 DNA polymerase, Taq DNA polymerase, T7 RNA polymerase and T4 polynucleotide kinase were purchased from Takara (Kyoto, Japan). Calf thymus terminal deoxynucleotidyl transferase and bovine pancreas deoxyribonuclease I were purchased from Stratagene Cloning Systems (La Jolla, CA). DNA polymerase assays. Activities of DNA polymerases were measured as described previously (12–14). Poly(dA)/oligo(dT) 12–18 and dTTP were used as template-primer DNA and nucleotide substrate for DNA polymerases, respectively. For HIV-1 reverse transcriptase, poly(rA)/oligo(dT) 12–18 and dTTP were used as template-primer and nucleotide substrate, respectively. For terminal deoxynucleotidyl transferase, oligo(dT) 12–18 (3⬘-OH) and dTTP were used as templateprimer and nucleotide substrate, respectively. One unit of each DNA polymerase activity was defined as the amount of enzyme that catalyzes the incorporation of 1 nmol of deoxyribonucleotide triphosphates (i.e., dTTP) into the synthetic template-primers (i.e., poly(dA)/oligo(dT) 12–18, A/T ⫽ 2/1) in 60 min at 37°C under the normal reaction conditions for each enzyme (12, 14). DNA topoisomerase assays. Relaxation activity of DNA topoisomerase II was determined by detecting the conversion of supercoiled pBR322 plasmid DNA to its relaxed form (15, 16). DNA topoisomerase II reaction was performed in 20 ␮l of reaction mixture containing 50 mM Tris-HCl buffer (pH 8.0), 120 mM KCl, 10 mM MgCl 2, 0.5 mM ATP, 0.5 mM dithiothreitol, pBR322 plasmid DNA (200 ng), 2 ␮l of inhibitor solution (10% DMSO) and 1 unit of DNA topoisomerase II. The reaction mixtures were incubated at 37°C for 30 min and reactions were terminated by adding 2 ␮l of loading buffer consisting of 5% sarkosyl, 0.0025% bromophenol blue and 25% glycerol. The mixtures were subjected to 1% agarose gel electrophoresis in TAE (Trisacetate-EDTA) running buffer. The agarose gel was stained with ethidium bromide and DNA was visualized on a UV transilluminator. One unit was defined as the amount of enzyme capable of relaxing 0.25 ␮g of DNA in 15 min at 37°C. Other enzyme assays. The activities of T7 RNA polymerase, T4 polynucleotide kinase and bovine deoxyribonuclease I were measured in each of the standard assays as reported by Nakayama et al. (17), Soltis et al. (18), and Lu and Sakaguchi (19), respectively.

RESULTS AND DISCUSSION Effects of Epolactaene on Various DNA Metabolic Enzymes Epolactaene was a epoxylactam ring and a long chain alkyl group. As shown in Fig. 1, we chemically synthesized epolactaene (compound 1) and its derivatives (compounds 2– 4). The chemical procedures will be published elsewhere, and this report is devoted to

FIG. 1. Structures of epolactaene and its derivatives. (A) Epolactaene (compound 1), (B) compound 2, (C) compound 3, and (D) compound 4.

analysis of the biochemical actions of the synthetic epolactaene and its derivatives in vitro. The derivatives of epolactaene all contained the epoxylactam ring but had straight long chain alkyl groups of different lengths (Figs. 1B–1D). Both compounds 2 and 4 had saturated straight chain alkyl groups (Figs. 1B and 1D). A significant portion of epolactaene-treated human SH-SY5Y cells exhibited a bipolar morphology, in which two neurites extended from opposite sides of the cell body in a dose-dependent manner (3). In addition, specific interactions between protease and protease inhibitors have been suggested to play a role in the regulation of neurite outgrowth (20, 21). However, epolactaene did not inhibit the activity of proteases such as papain, trypsin, cathepsin B and ␣-chymotrypsin in vitro, even at much higher concentrations than the effective dose on neurite outgrowth in cultured cells (6). As described in the introduction, epolactaene induced neurite outgrowth in human SH-SY5Y cells, and caused arrest of the cell cycle at G 0 /G 1 phase (6). Therefore, we investigated its inhibitory activities against DNA metabolic enzymes, because agents that arrest cell cycle influence proteins involved in DNA replication, repair and recombination. As shown in Fig. 2, epolactaene at 50 and 100 ␮M significantly inhibited the activities of both mammalian DNA polymerases (i.e., calf

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FIG. 2. Effects of epolactaene on the activities of various DNA polymerases and other DNA metabolic enzymes. Epolactaene (50 ␮M, gray bar and 100 ␮M, black bar) was incubated with each enzyme (0.05 units). The enzymatic activity was measured as described in previously (12–19). Enzyme activity in the absence of epolactaene was taken as 100%.

DNA polymerase ␣ and rat DNA polymerase ␤) and human DNA topoisomerase II. On the other hand, the activities of higher plant (cauliflower) DNA polymerase I (␣-like polymerase) and II (␤-like polymerase), prokaryotic DNA polymerases such as the Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase and Taq DNA polymerase, human immunodeficiency virus type-1 (HIV-1) reverse transcriptase, calf thymus terminal deoxynucleotidyl transferase, DNA metabolic enzymes such as T7 RNA polymerase, T4 polynucleotide kinase and bovine deoxyribonuclease I were not affected by epolactaene at this concentration (Fig. 1). Epolactaene should therefore be classified as a mammalian DNA polymerase-specific and/or human DNA topoisomerasespecific inhibitor.

were almost the same as not only those of aphidicolin and dideoxyTTP, well-known DNA polymerase ␣ and ␤ inhibitors, respectively (22, 23), but also those of other novel DNA polymerase inhibitors, the triterpenoids fomitellic acid A and B (24, 25), which induce neurite growth (26). These observations indicated that epolactaene is a potent inhibitor of mammalian DNA polymerases ␣ and ␤.

Effects of Epolactaene on the Activities of Mammalian DNA Polymerases ␣ and ␤ Figure 3 shows the inhibition dose-response curves of epolactaene on mammalian DNA polymerase ␣ and ␤. The inhibitory effect the compound was dosedependent, with 50% inhibition for DNA polymerase ␣ observed at a dose of 25 ␮M and almost complete inhibition (more than 80%) at 60 ␮M (Fig. 3). Fifty percent inhibition of DNA polymerase ␤ by epolactaene was obtained at 94 ␮M (Fig. 3). Epolactaene inhibited the activity of DNA polymerase ␣ to a greater extent than that of DNA polymerase ␤. The inhibitory doses

FIG. 3. Inhibition of mammalian DNA polymerase activities by epolactaene. Calf DNA polymerase ␣ (squares) and rat DNA polymerase ␤ (circles) (0.05 units each) were preincubated with the indicated concentrations (0 –100 ␮M) of epolactaene, and then enzymatic activities were assayed as described previously (12–14). DNA polymerase activity in the absence of epolactaene was taken as 100%.

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IC 50 Values of Epolactaene and Its Derivatives on the Activities of DNA Polymerases ␣ and ␤ and Topoisomerase II IC 50 value (␮M) Compound

DNA polymerase ␣

DNA polymerase ␤

DNA topoisomerase II

Epolactaene (Compound 1) Compound 2 Compound 3 Compound 4

25 26 ⬎200 ⬎200

94 98 ⬎200 ⬎200

10 20 ⬎200 ⬎200

Note. Epolactaene and its derivatives were incubated with each enzyme (0.05 units). The enzymatic activity was measured as described under Materials and Methods. Enzyme activity in the absence of the compounds was taken as 100%.

Effects of Epolactaene on Human DNA Topoisomerase II As shown in Table 1, epolactaene (compound 1) and compound 2 inhibited the activities of both mammalian DNA polymerases and human DNA topoisomerase II. The inhibitory effect of epolactaene on DNA polymerase ␣ was almost same as that on DNA topoisomerase II. When SH-SY5Y cells were treated with epolactaene at 6.25 to 25 ␮M, many neurites extended from the cell bodies in a dose-dependent manner (3). At this concentration range, epolactaene weakly inhibited the activities of DNA polymerases and topoisomerase II (Table 1 and Fig. 3). On the other hand, compounds 3 and 4 required concentrations higher than 200 ␮M for 50% inhibition. Epolactaene showed almost the same inhibitory effects against these enzymes as compound 2 (Table 1). The results indicated that the long chain alkyl group of the molecule is necessary for the inhibitory effect on these enzymes. To test whether epolactaene is an intercalating agent that distorts DNA and subsequently inhibits the enzyme activities, we measured thermal transition of DNA in the presence or absence of epolactaene. The thermal transition profiles of DNA with or without epolactaene were unchanged (data not shown). Therefore, the inhibition of both DNA polymerases and topoisomerase II by epolactaene was not due to DNA distortion, but appeared to be due to a direct effect of this compound on the enzyme proteins themselves. Epolactaene is a neurotrophic factor involved in survival, growth and differentiation of neoplastic nerve cells (1, 2). Kakeya et al. (6) reported that in the human neuroblastoma cell line, SH-SY5Y, treated with epolactaene, the fraction of cells at S phase decreased, while the fraction in G 0/G 1 phase increased, suggesting that the compound significantly arrested the cell cycle at G 0/G 1 phase. On the other hand, the lack of replicative DNA polymerase and DNA topoisomerase was reported to inhibit cell cycle progression at G 1/S or G 0/G 1 phase (7, 8), although the role of DNA polymerase ␤ in cell cycle progression is still unclear. DNA polymerases ␣ and ␤ and DNA topoisomerase II might be target

molecules for epolactaene. Since epolactaene is a nonprotein neurotrophic compound that induces neurite outgrowth in human SH-SY5Y cells lacking significant TRK family mRNA (4, 5), this compound may be a good candidate for pharmaceutical use to study various neurodegenerative diseases. The relationship between neuritogenesis and cell cycle arrest should be clarified using epolactaene, and subsequent studies of the inhibition of DNA polymerases ␣ and ␤ and DNA topoisomerase II should also clarify the molecular mechanism of neuronal differentiation. Selection of whether cells at G 1 phase enter into S phase or G 0 phase may be a key for neurodegeneration or neuronal differentiation, respectively. Based on the present results, we speculated that selection at G 1 phase might be triggered by modifying the structure and function of DNA polymerases ␣ and ␤ and/or DNA topoisomerase II, and inhibition of these enzymes at G 1 might turn the G 1 cells toward G 0 phase, and subsequently they may undergo neuritogenesis. ACKNOWLEDGMENTS We are grateful to Dr. S. Yoshida of Nagoya University and Dr. A. Matsukage of Japan Women’s University for preparing calf DNA polymerase ␣ and rat DNA polymerase ␤, respectively, and for valuable discussions about the inhibitors. This work was supported in part by grants to Y.M. from the Mochida Memorial Foundation for Medical and Pharmaceutical Research and the Uehara Memorial Foundation.

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