The effects of dehydroaltenusin, a novel mammalian DNA polymerase α inhibitor, on cell proliferation and cell cycle progression

Biochimica et Biophysica Acta 1674 (2004) 193 – 199 www.bba-direct.com

The effects of dehydroaltenusin, a novel mammalian DNA polymerase a inhibitor, on cell proliferation and cell cycle progression Chikako Murakami-Nakaia, Naoki Maedaa, Yuko Yonezawaa, Isoko Kuriyamaa, Shinji Kamisukib, Shunya Takahashic, Fumio Sugawarab, Hiromi Yoshidaa,d, Kengo Sakaguchib, Yoshiyuki Mizushinaa,d,* a

Laboratory of Food and Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe 651-2180, Hyogo, Japan b Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan c Synthetic Organic Chemistry Laboratory, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-9800, Japan d High Technology Research Center, Kobe-Gakuin University, Nishi-ku, Kobe 651-2180, Hyogo, Japan Received 10 March 2004; received in revised form 15 June 2004; accepted 16 June 2004 Available online 8 July 2004

Abstract As described previously, a natural product isolated from fungus (Acremonium sp.), dehydroaltenusin, is an inhibitor of mammalian DNA polymerase a in vitro [Y. Mizushina, S. Kamisuki, T. Mizuno, M. Takemura, H. Asahara, S. Linn, T. Yamaguchi, A. Matsukage, F. Hanaoka, S. Yoshida, M. Saneyoshi, F. Sugawara, K. Sakaguchi, Dehydroaltenusin, a mammalian DNA polymerase a inhibitor, J. Biol. Chem. 275 (2000) 33957_33961]. In this study, we investigated the interaction of dehydroaltenusin with lipid bilayers using an in vitro liposome system, which is a model of the cell membrane, and found that approximately 4% of dehydroaltenusin was incorporated into liposomes. We also investigated the influence of dehydroaltenusin on cultured cancer cells. Dehydroaltenusin inhibited the growth of HeLa cells with an LD50 value of 38 AM, and as expected, S phase accumulation in the cell cycle. The total DNA polymerase activity of the extract of incubated cells with dehydroaltenusin was 23% lower than that of nontreated cells. Dehydroaltenusin increased cyclin E and cyclin A levels. In the analysis of the cell cycle using G1/S synchronized cells by employing hydroxyurea, the compound delayed both entry into the S phase and S phase progression. In a similar analysis using G2/M synchronized cells by employing nocodazole, the compound accumulated the cells at G1/S and inhibited entry into the S phase. Thus, the pharmacological abrogation of cell proliferation by dehydroaltenusin may prove to be an effective chemotherapeutic agent against tumors. D 2004 Elsevier B.V. All rights reserved. Keywords: Dehydroaltenusin; DNA polymerase a; Enzyme inhibitor; Lipid bilayers of liposomes; Human cancer cell; Cell growth inhibition; Cell cycle arrest

1. Introduction Abbreviations: pol, DNA polymerase (EC 2.7.7.7); dNTP, deoxyribonucleoside triphosphate; egg PC, phosphatidyl choline from egg yolk; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; MEM, Eagle’s minimum essential medium; DMSO, dimethylsulfoxide; DAPI, 4V6-diamidino-2-phenylindole; PVDF, polyvinyldenefluoride; PCNA, proliferating cell nuclear antigen; HU, hydroxyurea; Noc, nocodazole * Corresponding author. Laboratory of Food and Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe 651-2180, Hyogo, Japan. Tel.: +81 78 974 1551x3232; fax: +81 78 974 5689. E-mail address: [email protected] (Y. Mizushina). 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.06.016

Eukaryotic multiple organisms are known to contain at least 14 types of DNA polymerases [1,2]. We screened for natural compounds that were selective inhibitors of these enzymes [3–6]. Selective inhibitors of DNA polymerases are useful tools in distinguishing DNA polymerases and clarifying their biological and in vivo functions [7]. Inhibition by the well-known replicative DNA polymerase inhibitor aphidicolin demonstrated that DNA polymerase a, y and q (pol a, y and q) are essential for replication [8].

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Aphidicolin has been very useful in studying the DNA replication system [9], however, there have been no previous reports of inhibitors capable of distinguishing among pol a, y and q. Among the three DNA polymerases, pol a is not only essential for DNA replication, but is also involved in cell division [1]. Selective inhibitors of mammalian pol a are not only molecular tools useful for analyzing polymerases, but should also be considered a group of potentially useful cancer chemotherapy agents. Dehydroaltenusin (Fig. 1) was isolated from a fungus (Acremonium sp.) in the screening for replicative DNA polymerase inhibitors [10]. It was found to be a strong inhibitor of mammalian pol a, having an IC50 value of 0.68 AM, but it did not influence the activities of mammalian pol y and q, nor pol a from other vertebrates in vitro [10]. In this report, we measured the amounts of dehydroaltenusin incorporated into the lipid bilayers of liposomes to estimate their affinities for cell membranes. An accurate method to measure the amount of DNA polymerase inhibitors incorporated into a model membrane is needed. In the pharmaceutical sciences, liposome partitioning systems have been used to investigate the relationship between the biological activity of compounds and their interaction with lipid bilayers [11,12]. However, there have been no reports on the affinity of the compounds for model membranes. One problem is the difficulty of separating the liposomes from the aqueous medium after incubation of the compounds. To solve this problem, we performed a method whereby we separated the medium and the liposomes with a dense internal aqueous phase using centrifugation. We found that dehydroaltenusin could be incorporated into liposomes and could prevent the proliferation of human cultured cancer cells by halting the cell cycle. This suggests that dehydroaltenusin could penetrate into cells and then influence DNA synthesis. Based on the analysis of the details of the cell cycle arrest mechanisms and growth inhibition, we suggest such an action of pol a inhibition.

Fig. 1. Structure of dehydroaltenusin.

2. Materials and methods 2.1. Materials Dehydroaltenusin (Fig. 1) was chemically synthesized and supplied as described previously [13]. Phosphatidyl choline from egg yolk (egg PC) was purchased from Wako Pure Chemical Ind. Ltd. (Tokyo, Japan). A human cervix carcinoma cell line, HeLa, was obtained from Health Science Research Bank (Osaka, Japan). All other reagents were of analytical grade and were purchased from Nacalai Tesque Inc. (Kyoto, Japan). 2.2. Measurement of DNA polymerase activity HeLa cells were plated at 3105 into a 100-mm culture dish with or without 38 AM of dehydroaltenusin. After zero, 6 and 12 h of incubation, the cells were washed with phosphate buffered saline (PBS), collected by centrifugation, and then their pellets were sonicated for 3 min in a 50 mM Tris–HCl (pH 7.5) buffer containing 1 mM EDTA, 5 mM 2-mercaptoethanol, 15% glycerol, 1 Ag/ml leupeptin and 100 AM phenylmethylsulfonyl fluoride (PMSF). The DNA polymerase activity of the cell extract was assayed as described previously [3,4]. One unit of enzyme activity was defined as the amount that catalyzes the incorporation of 1 nmol of dexyribonucleoside triphosphates (i.e., dTTP) into synthetic templateprimers (i.e., poly(dA)/oligo(dT)12–18, A/T=2:1) at 37 8C in 60 min. 2.3. Measurement of dehydroaltenusin incorporated into lipid bilayers The affinity of dehydroaltenusin for liposomes, a model of a cell membrane, was measured as reported by Nakayama et al. [14]. A thin film of egg PC on the inner surface of a flask was dried with a vacuum pump. A 300 mM aqueous glucose solution was then poured into the flask, and the mixture was sonicated in a bath type sonicator. The resulting solution of multilamellar vesicles was then sonicated in a cup-horn type of sonicator to change the multilamellar vesicles into small unilamellar vesicles. The liposomal solution was diluted 10-fold with PBS. The final concentration of egg PC in the liposomal solution was adjusted to 1 mg/ml. Various concentrations of dehydroaltenusin solution in PBS were added to the liposomal solutions. After incubation for 20 min at 37 8C, the mixture was centrifuged at 130,000g for 5 min at room temperature. After being suspended with PBS and centrifuged again, the liposome pellets containing the dehydroaltenusin were dissolved with 1 ml of ethanol. The amount of dehydroaltenusin in the ethanol solution was measured using a UV-spectophotometer (UV-2500PC, Shimadzu Co., Kyoto, Japan), with a detection wavelength of 298.8 nm (dehydroaltenusin,

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UV k max (EtOH) nm (e): 298.8 (8650)). The percentage of dehydroaltenusin incorporated into the lipid bilayers was calculated as follows: [amount incorporated]/[amount added]100.

3. Results and discussion

2.4. Cell culture and measurement of cell viability

To determine the effects of dehydroaltenusin in cultured cancer cells, we tested their influence on HeLa cell growth, a human cervix carcinoma cell line. Dehydroaltenusin efficiently inhibited cell growth in a dose-dependent manner, and the LD50 value was 38 AM for 12-h incubation (Fig. 2). NUGC-3 cells (human stomach cancer cell line) and A-549 cells (human lung cancer cell line) were also inhibited the cell growth by dehydroaltenusin with the LD50 values of 43 and 44 AM, respectively. HeLa, NUGC-3 and A-549 cells are adherent cell lines. BALL-1 cells (human acute lymphoblastoid leukemia cell line) as a nonadherent cell line was suppressed with almost the same cell growth inhibitory results as the HeLa cells (Fig. 2). Therefore, dehydroaltenusin might have a generalized observed effect on bhuman cancer cellsQ. The total DNA polymerase activity of the dehydroaltenusin treated cell extract was lower than that of nontreated cells. Enzyme activity was decreased 23.4% after 6 h and 21.3% after 12 h (Fig. 3). Since dehydroaltenusin specifically inhibited only the activity of pol a, it is possible to predict that approximately 23% of the total DNA polymerase activity in HeLa cells arises from pol a. We would, however, like to emphasize here that dehydroaltenusin at first intercalates into the DNA molecule as the substrate (i.e., DNA template-primer), and subsequently inhibits

HeLa cells were cultured in Eagle’s minimum essential medium (MEM) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 mg/ml) and 1% dimethylsulfoxide (DMSO) at 37 8C in a humid atmosphere of 5% CO2/95% air. For the cell viability assay, cells were plated at 3105 into each well of 96-well microplates with various concentrations of dehydroaltenusin. Dehydroaltenusin was dissolved in DMSO at a concentration of 10 mM as a stock solution. The stock solution was diluted to the appropriate final concentrations with growth medium just before use. The cell viability was determined using MTT assay [15]. 2.5. Cell cycle analysis Cells (3105 cells in 35 mm dish) were collected via trypsinization and washed with ice-cold PBS by centrifugation. The cells were suspended in PBS, fixed with 70% ethanol (v/v), and stored at 20 8C. The cells were collected via centrifugation and stained with 4V-6-diamidino-2-phenylindole (DAPI) (2 Ag/ml) for at least 20 min at room temperature in the dark. The DNA content of the 8000 stained cells was analyzed using a cell counter analyzer (Partec, CCA Model; Munster, Germany) with Multicycle 3.11 software (Phoenix Flow Systems, San Diego, CA). The cell debris and fixation artifacts were gated out.

3.1. Effect of dehydroaltenusin on cancer cell growth inhibition and DNA polymerase activity

2.6. Western blot analysis The cells were lysed in RIPA buffer (50 mM Tris–HCl (pH 7.2), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.05% sodium dodecyl sulfate (SDS), 1 mM PMSF and 1 mM leupeptin). Cell lysates were centrifuged at 14,000g at 4 8C for 10 min. The supernatant was analyzed by SDS-PAGE. To extract the protein binding chromatin, cells were lysed with cold 0.1% Triton X-100 in CSK buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 300 mM sucrose, 1 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, and 1 mM PMSF) for 20 min at 4 8C. Triton-insoluble fractions were solubilized in CSK buffer with DNase I (0.5 mg/ml DNase I) for 30 min. The samples were centrifuged at 16,000g for 15 min to obtain the supernatant. Equal amounts of protein were separated using SDS-PAGE, then blotted on a polyvinyldenefluoride (PVDF) membrane. The blots were subsequently incubated with the desired primary antibody. A Zero-D scan was used for densitometric quantitation.

Fig. 2. Effect of dehydroaltenusin on the cell proliferation of human cancer cells. Dose-dependent inhibitions of the lung cancer cell line, A549 (open circle), stomach cancer cell line, NUGC-3 (closed diamond), cervix cancer cell line, HeLa (closed triangle) and acute lymphoblastoid leukemia cell line, BALL-1 (open square) incubated with various concentrations of dehydroaltenusin for 12 h. Cell proliferation was determined by MTT assay [15]. Values are shown as meansFS.E. for four independent experiments.

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3.2. Dehydroaltenusin incorporation into liposomes Fig. 4 shows the typical dose effects of dehydroaltenusin incorporation into liposomes of egg yolk phosphatidyl choline (egg PC). The amount incorporated increased as the amount of the compound added to the liposomal solution in the range tested increased. This result suggests that the amount incorporated reflected the affinities of dehydroaltenusin for the lipid bilayers of liposomes. The proportion of

Fig. 3. Total DNA polymerase activity of cell-free extracts from HeLa cells incubated with or without dehydroaltenusin. HeLa cells (3105 cells) were incubated with or without 38 AM of dehydroaltenusin for zero, 6 and 12 h. From the cell extracts the total DNA polymerase activity was measured as described previously [3,4]. One unit of DNA polymerase activity is defined as the amount that catalyzes the incorporation of 1 nmol of dexyribonucleoside triphosphates (i.e., dTTP) into synthetic template-primers (i.e., poly(dA)/oligo(dT)12–18, A/T=2:1) at 37 8C in 60 min. Values are shown as meansFS.E. for four independent experiments.

both activities indirectly through the induction of a conformational change in the DNA. This compound effected no thermal transition of melting temperature (data not shown); thus, none of dehydroaltenusin bound to the dsDNA, suggesting that this must inhibit the enzyme activities by interacting with pol a directly. We then investigated whether an excessive amount of poly(rC) or BSA could prevent the inhibitory effect of dehydroaltenusin to determine whether the effect resulted from their nonspecific adhesion to pol a or selective binding to specific sites. Poly(rC) and BSA had little or no influence on the effect of dehydroaltenusin, suggesting that the binding to pol a occurs selectively.

Fig. 4. Dose effects of dehydroaltenusin on the amount incorporated into the liposomes. Dehydroaltenusin was added to liposomal solution and the mixture was incubated for 20 min at 37 8C. After centrifugation of the mixture, the amount of dehydroaltenusin incorporated into the liposomes was measured using UV absorption (298.8 nm).

Fig. 5. The effect of dehydroaltenusin on the cell cycle. (A) Flow cytometric analysis of HeLa cells treated with 38 AM dehydroaltenusin. The cell cycle distribution was calculated as the percentage of cells that were in the G1, S and G2/M phase. (B) Cyclin expressions were analyzed using Western blotting. Cell extracts of the nuclear fraction were prepared from cells treated with 38 AM dehydroaltenusin. Cyclin E, A and B were detected with specific antibodies. Densitometric assay of the proteins was performed and fold induction was calculated.

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percentage of cells in the G2/M phase decreased (30.6% to 17.3%). G1 phase cells were not changed through incubation (30.2% to 32.6%). We examined whether the cell cycle

Fig. 6. The effect of dehydroaltenusin on the binding of the DNA polymerase complex to chromatin. Western blot analysis of pol a, pol q and PCNA in the chromatin-bound fraction.

the incorporated dehydroaltenusin for 20-min incubation was approximately 4%, and this percentage was almost the same value of caffeic acid incorporation into the liposomes [14]. Since the percentage for 24-h incubation of liposome solution with dehydroaltenusin was the same value (i.e., about 4%) as 20-min incubation (data not shown), the incubation time may not reflect incorporation time. The biological activities of many natural and synthetic DNA polymerase inhibitors have been examined using various methods in vitro and in vivo to predict their ability to prevent cancer. The activity found in in vivo experiments with cultured mammalian cells reflects two main factors: The first is real activity, with the assumption that the same amount of every compound is incorporated into the cells. The second is the amount actually incorporated during incubation. If the assumption about real activity is correct, then the amount actually incorporated governs the degree of biological activity. In other words, even if the concentration of a compound in a medium is low, efficient incorporation would generate high activity. As shown in Fig. 4, the amount of dehydroaltenusin incorporated into the liposomes was low, therefore, the pol a inhibitory activity caused by incorporating compounds into cells might have been high (the IC50 value for pol a activity is 0.68 AM [10]). When 38 AM of dehydroaltenusin was added to the cell culture medium (Fig. 2), approximately 1.5 AM of dehydroaltenusin was thought to have penetrated and/or been incorporated into HeLa cells as a result of the incorporation of dehydroaltenusin into the lipid bilayer of liposomes which is a model of a cell membrane. Since dehydroaltenusin is a strong mammalian pol a specific inhibitor, the incorporated compound (i.e., the concentration is 1.5 AM) could completely inhibit pol a activity in the cell. 3.3. Effect of dehydroaltenusin on the cancer cell cycle To clarify the effect of dehydroaltenusin on cell cycle regulation, we analyzed the cell cycle using flow cytometry with DAPI staining (Fig. 5A). When the cells were treated with 38 AM (=LD50 value) dehydroaltenusin, the distribution of cells in the cell cycle changed time-dependently. In the presence of dehydroaltenusin for 12 h, the percentage of cells in the S phase increased (39.1% to 50.1%) and the

Fig. 7. Effect of dehydroaltenusin on cell cycle progression in synchronized cells. (A) Cells were treated with 2 mM HU or 25 AM Noc for 12 h, after which cells were washed in fresh medium and treated with or without 38 AM dehydroaltenusin. (B) Cells were synchronized in G1/S phase using HU. Cells were released into cycle with or without 38 AM dehydroaltenusin for the indicated times. (C) Cells were synchronized in G2/M phase using Noc. Cells were released into cycle with or without 38 AM dehydroaltenusin for the indicated times.

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effect of dehydroaltenusin was associated with the expression of cyclin proteins using Western blotting (Fig. 5B). Cyclin A and cyclin E proteins which are regulated in the G1/S phase [16–19] increased with dehydroaltenusin treatment, but cyclin B which is regulated in the G2/M phase [20] decreased significantly. These results suggest that dehydroaltenusin induced S phase arrest in human cancer cells. 3.4. Effect of replication delay Since dehydroaltenusin inhibited mammalian pol a activity in vitro [10], we hypothesized that the growth inhibition by dehydroaltenusin was a result of its pol a inhibition. To test this possibility, the presence of pol a, proliferating cell nuclear antigen (PCNA) and pol q were detected using Western blots. After treatment with 38 AM of dehydroaltenusin at various times, protein expression levels were assessed (Fig. 6). The protein expression patterns were consistent with the cell cycle analysis data. An increase of the cells in the S phase occurred accompanied with PCNA accumulation on the chromatin fraction. But pol a and pol q did not change through incubation with dehydroaltenusin. Aphidicolin, an inhibitor of pol a, y and q [21], inhibited DNA chain elongation completely by competing with each the four dNTPs for binding to a pol a–DNA binary complex but not on the primary initiation event of primase activity [22]. From this characteristic, aphidicolin acts as synchronization of cell cycle at G1/S phase and S-phase progression followed on removing the drug [23]. We reported previously that dehydroaltenusin did not influence the primase activity of the smallest subunit of pol a, and the activities of pol y and pol q [10]. These results suggest that cell growth inhibition caused by dehydroaltenusin resulted from DNA replication inhibition in the middle S phase, not complete inhibition at G1/S phase, because DNA synthesis caused by the deoxyribonucleoside triphosphate (dNTP)-polymerization activity of the largest subunit of pol a did not progress efficiently. We reported previously that dehydroaltenusin did not influence the primase activity of the smallest subunit of pol a [10]. These results suggest that cell growth inhibition caused by dehydroaltenusin resulted from DNA replication inhibition in the middle S phase because DNA synthesis caused by the dNTP-polymerization activity of the largest subunit of pol a did not progress efficiently.

dehydroaltenusin induced an S phase delay. To discriminate between these two possibilities, cells were treated with hydroxyurea (HU), which is a ribonucleotide reductase inhibitor [25–27], and nocodazole (Noc), which is an inhibitor of microtubule polymerization [28–30], before cells were treated with dehydroaltenusin, respectively. The treatment schedule is shown in Fig. 7A. A G1/S synchronization with HU showed that both entry into the S phase and cell cycle progression of the S phase were delayed compared to control cells (Fig. 7B). However, aphidicolin inhibited entry into the S phase completely (data not shown). On the other hand, Noc synchronization, referring to a large number of cells that were in G2/M, showed that dehydroaltenusin also delayed the progression to S phase because the middle S phase appearance was suppressed under dehydroaltenusin treatment (Fig. 7C). These results indicated a DNA replication delay because of dehydroaltenusin treatment. Under this condition, it was estimated that dehydroaltenusin delayed replication without DNA damage because p53, bax and bcl-2 protein expression levels did not change and DNA fragmentation did not occur (data not shown). However, DNA fragmentation was detected in the cells which were treated with higher concentration of dehydroaltenusin (data not shown). These results suggest that the moderate inhibition of pol a activity induced only replication delay, but the strong inhibition of pol a activity by the compound led to the cytogenetic instability. Because dehydroaltenusin selectively inhibits pol a activity, it can possibility be used as an anti-cancer chemotherapeutic agent yielding genomic instability to cancer cells.

Acknowledgements This work was partly supported by a Grant-in-Aid for Kobe Gakuin University Joint Research (B) (H.Y. and Y.M.) and the Chemical Biology Project (RIKEN) (S.T.). Y.M. acknowledges Grants-in-Aid from the Hyogo Science and Technology Association, The Japan Food Chemical Research Foundation and Grant-in-Aid 16710161 for Scientific Research, The Ministry of Education, Culture, Sports, Science and Technology, Japan.

References 3.5. Dehydroaltenusin inhibition and delayed S-phase progression A replicative DNA polymerase inhibitor, aphidicolin, arrested cell cycle at the G1/S phase [24]. As shown in Fig. 5A, the population of the cells in the S phase increased when the cells were treated with dehydroaltenusin. Two possibilities may account for the S phase accumulation caused by dehydroaltenusin. One is that dehydroaltenusin blocked cell entry into the S phase, and the other is that

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