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The Histone Deacetylase Inhibitor Sodium Butyrate Induces DNA Topoisomerase II␣ Expression and Confers Hypersensitivity to Etoposide in Human Leukemic Cell Lines1

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The Histone Deacetylase Inhibitor Sodium Butyrate Induces DNA Topoisomerase II␣ Expression and Confers Hypersensitivity to Etoposide in Human Leukemic Cell Lines1 Vol. 1, 121–131, December 2001 Molecular Cancer Therapeutics 121 The Histone Deacetylase Inhibitor Sodium Butyrate Induces DNA Topoisomerase II␣ Expression and Confers Hypersensitivity to Etoposide in Human Leukemic Cell Lines1 Ebba U. Kurz,2 Sara E. Wilson,3 Kelly B. Leader, stimulated, protein-linked DNA complexes in both cell Brante P. Sampey, William P. Allan, lines. At concentrations with minimal effects on cell 4, 5 Jack C. Yalowich, and David J. Kroll cycle kinetics (0.4 mM in HL-60; 0.5 mM in K562), NaB Department of Pharmaceutical Sciences [E. U. K., S. E. W., K. B. L., pretreatment also modestly enhanced etoposide- B. P. S., D. J. K.], Center for Pharmaceutical Biotechnology [D. J. K.], triggered apoptosis in HL-60 cells, as determined and Program in Molecular Toxicology and Environmental Health Sciences [B. P. S., D. K. J.], School of Pharmacy, University of morphologically after acridine orange/ethidium Colorado Health Sciences Center, and University of Colorado Cancer bromide staining, and substantially increased K562 Center [E. U. K., D. J. K.], Denver, Colorado 80262, and Department of Pharmacology, University of Pittsburgh School of Medicine and Cancer growth inhibition and poly(ADP-ribose)polymerase Institute, Pittsburgh, Pennsylvania 15261 [W. P. A., J. C. Y.] cleavage after etoposide exposure. Therefore, a temporal window may exist whereby a differentiating agent may sensitize experimental leukemias to a Abstract cytotoxic antitumor agent. These results indicate that The differentiating agent and histone deacetylase histone deacetylase inhibitors should be investigated inhibitor, sodium butyrate (NaB), was shown previously for etoposide sensitization of other butyrate-responsive to cause a transient, 3–17-fold induction of human DNA hematopoietic and nonhematopoietic tumor lines in topoisomerase II␣ (topo II␣) gene promoter activity and vitro and in vivo. a 2-fold increase in topo II␣ protein early in monocytic differentiation of HL-60 cells. This observation has now Introduction been extended to other short chain fatty acids and The transcriptional and posttranscriptional regulation of DNA aromatic butyrate analogues, and evidence is topo6 II␣, a nuclear enzyme essential for completion of mi- presented that human topo II␣ promoter induction tosis (1–3), has been a primary focus of our research groups correlates closely with histone H4 acetylation status. (4–8). Drugs such as the epipodophyllotoxins (etoposide and Because increased topo II␣ expression is associated teniposide), anthracyclines, ellipticines, anthracenediones, with enhanced efficacy of topo II-poisoning antitumor and aminoacridine derivatives exert their cytotoxic activity, at drugs such as etoposide, the hypothesis tested in this least in part, by trapping topo II in a covalent complex with report was whether NaB pretreatment could sensitize DNA (9). These “cleavable complexes” (10) can act as phys- HL-60 myeloid leukemia and K562 erythroleukemia ical barriers to DNA replication (11–13), cause mitotic catas- cells to etoposide-triggered DNA damage and cell trophes (14), induce recombination events (15, 16), and/or death. A 24–72 h NaB treatment (0.4–0.5 mM) induced trigger a cytotoxicity cascade culminating in apoptosis (17– topo II␣ 2–2.5-fold in both HL-60 and K562 cells and 19). Because the class of antitumor drugs converts this es- caused a dose-dependent enhancement of etoposide- sential enzyme into a lethal instrument, it follows that tumor cell populations possessing high levels of topo II␣ are more effectively killed by these agents. Received 8/13/01; revised 9/19/01; accepted 9/25/01. Two genes exist for mammalian type II DNA topoisomer- The costs of publication of this article were defrayed in part by the ases, termed ␣ and ␤, likely attributable to evolutionary gene payment of page charges. This article must therefore be hereby marked ␣ advertisement in accordance with 18 U.S.C. Section 1734 solely to indi- duplication (20, 21). topo II is often the more predominant cate this fact. cellular form (22, 23). Cleavable complexes formed with topo 1 This work was supported by Grant RPG-97-084-01-CDD from the Amer- II␣ correlate more closely with teniposide cytotoxicity in leu- ican Cancer Society, a grant from the Cancer League of Colorado (both to ␤ D. J. K.), NIH Grant CA74972 (to J. C. Y.), and an American Cancer So- kemia cells than do those formed with topo II (24), but in ciety/Brooks Trust Fellowship Award (to S. E. W.). The experiments from other systems topo II␤ may also influence drug sensitivity the University of Colorado Cancer Center Flow Cytometry Core Facility (25, 26). The ␣ form is expressed in a proliferation-dependent were supported by NIH Grant P30 CA46934 from the National Cancer Institute. manner, whereas the ␤ form is expressed independently of 2 Present address: Department of Biological Sciences, University of Cal- growth status (27–29). Therefore, rapidly growing cells that gary, Calgary, Alberta, T2N 1N4 Canada. 3 Present address: Department of Biological Sciences, Stanford Univer- sity, Stanford, CA 94305. 4 Present address: Division of General Internal Medicine, Department of Medicine, Duke University Medical Center, Duke Comprehensive Cancer 6 The abbreviations used are: topo, topoisomerase; NaB, sodium butyr- Center, Durham, NC 27710. ate; HDAC, histone deacetylase; IC50, drug concentration that inhibits 5 To whom requests for reprints should be addressed, at Duke University growth by 50%; PARP, poly(ADP-ribose)polymerase; ATCC, American Medical Center, Box 3020, Microbiology, Durham, NC 27710. Phone: Type Culture Collection; CMV, cytomegalovirus; ␤-gal, ␤-galactosidase; (919) 668-0281; Fax: (919) 309-4042; E-mail: [email protected]. SCFA, short-chain fatty acid; TSA, trichostatin A. Downloaded from mct.aacrjournals.org on October 2, 2021. © 2001 American Association for Cancer Research. 122 HDAC Inhibitors and topo II␣ Induction contain high topo II␣ levels are effectively killed by the topo and sodium phenylbutyrate manufactured by Elan Pharma- II-directed drugs, whereas differentiated or otherwise ceuticals (San Francisco, CA) were generous gifts of Dr. growth-arrested cells usually possess diminished topo II␣ Andrew Kraft (Division of Medical Oncology, University of levels and are intrinsically resistant to these agents (9, 30, Colorado School of Medicine). Buffers and all other chemi- 31). Similarly, acquired resistance to a topo II-directed agent cals were obtained from United States Biochemical/Amer- is often attributable to suppressed topo II␣ and/or topo II␤ sham Pharmacia Biotech (Arlington Heights, IL). Chemicals expression (8, 32, 33). were of the highest purity listed by the supplier, and molec- Several transcription factors have been linked to the high, ular biology-tested reagents were obtained wherever pos- proliferation-dependent expression of topo II␣ including c- sible. Myb and B-Myb (5), NF-M (4), NF-Y (34–37), and YB-1 (38). Mammalian Cell Culture. HL-60 human promyelocytic The Sp3 transcription factor is also known to activate the cells (ATCC CCL 240) or K562 human erythroleukemia cells ␣ human topo II gene (39). Conversely, the p53 tumor sup- (ATCC CCL 243) were obtained from the ATCC (Manassas, ␣ pressor protein represses the human topo II gene through VA). Cells were maintained as suspension cultures in either the basal transcriptional machinery (40) or via an inverted RPMI 1640 (HL-60) or Iscove’s modified Minimal Essential Ϫ CCAAT box at position 68 (41). The activated Ras pathway Medium (K562; Life Technologies, Inc.) supplemented with ␣ ␣ also stimulates both topo II activity and expression. topo II 10% fetal bovine serum, 50 units/ml penicillin G, and 50 activity is enhanced via protein interactions with extracellular ␮g/ml streptomycin sulfate. All cells were maintained at 37°C ␣ signal-regulated kinase 2 (42), and topo II trans-activation in a humidified atmosphere containing 5% CO . Cells were can be driven through a Ets-like enhancer element at position 2 carried as exponentially growing cultures by propagation at Ϫ480 of the topo II␣ promoter (43). Finally, hyperthermia also 5 ϫ 105 cells/ml every 2–3 days. induces topo II␣ expression in vitro (44) as a result of in- Cellular Histone Acetylation Assay. Nuclei were iso- creased topo II␣ mRNA stability (45). lated, and histone fractions were prepared from cell nuclei as In previous studies, the serendipitous observation was described (50) with the following modifications. Pelleted nu- made that topo II␣ transcription and gene expression was clei were resuspended in 1.35 ml of sterile, ice-cold water transiently induced by the monocytic differentiating agent, containing 1 mM phenylmethylsulfonyl fluoride and 0.15 ml of NaB (6). NaB has long been recognized as an inhibitor of 4 N H SO added dropwise with swirling and histones ex- HDAC activity (46), an effect that is, in turn, associated with 2 4 tracted overnight. After centrifugation at 12,000 ϫ g for 10 selective gene activation or repression (47–49). It was hy- min, histone supernatants were transferred to 15 ml of Fal- pothesized that as a result of increased topo II␣ expression, con polypropylene tubes and filled with cold acetone/HCl NaB-pretreated leukemia cells would exhibit increased sen- Ϫ sitivity to etoposide because of increased topo II-mediated (99:1 acetone:5 N HCl) and incubated at 20°C for 72 h. DNA damage. During the final pelleting of the histone isolates, all samples ϫ In the present study, we show that several SCFAs and were initially centrifuged
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