[CANCER RESEARCH 63, 8955– 8961, December 15, 2003] Butyrates, as a Single Drug, Induce Histone Acetylation and Granulocytic Maturation: Possible Selectivity on Core Binding Factor-Acute Myeloid Leukemia Blasts Antonella Gozzini, 1 Elisabetta Rovida, 2 Persio Dello Sbarba, 2 Sara Galimbert, 3 and Valeria Santini 1 1 Departments of Hematology and 2 Experimental Pathology and Oncology, Universita ` di Firenze, Florence, Italy and 3 Department of Hematology, Universita ` di Pisa, Pisa, Italy ABSTRACT Acute myeloid leukemia (AML) is a disease characterized by a block of maturation. Genes coding for core binding factors are rearranged in a considerable subset of AML cases and result in an altered interaction of core binding factor (CBF) subunits with transcriptional coregulators (NCoR/SMRT). Recruitment of histone deacetylase is also altered in AML, and a subsequent transcriptional repression of target genes in- volved in myeloid maturation is determined. We determined here the effects of two histone deacetylase inhibitors, sodium butyrate and the stable prodrug xylitol butyrate derivative (D1), on a t(8;21)-positive cell line (Kasumi-1) as well as primary AML blasts. Exposure (24 –96 h) to butyrates (1 mM) of Kasumi-1 cells induced histone H4 acetylation, whereas H3 acetylation was unchanged. Induction of morphological and immunophenotypic granulocytic maturation (96 h), also confirmed by an increased expression of CAAT/enhancer binding protein , was observed. Inhibition of proliferation and apoptosis via activation of caspase-9 was also observed. In primary AML blasts, butyrates (0.5 mM) increased histone H4 acetylation of 18 of 19 cases tested. Terminal granulocytic maturation was observed in all cases (5 of 5) characterized by chromo- somal translocations involving CBF, whereas in non-CBF cases, matura- tion was incomplete (4 of 8) or absent (4 of 8). Our data indicate the possibility to effectively remove, in CBF AML cases, the maturation block generated by histone deacetylase stable recruitment, contributing to a possible development of molecularly targeted therapies of AML. INTRODUCTION Acute myeloid leukemia (AML) is biologically a highly heteroge- neous disease. Multiple recurrent chromosome and gene rearrange- ments have been identified and contribute to delineation of prognos- tically distinct categories of AML (1). One such category has been defined recently as core binding factor (CBF) AML. Patients with CBF AML (whose cells most commonly exhibit either t(8;21)(q22; q22) AML1/ETO or inv(16)(p13q22) CBF/MYH11) constitute ap- proximately 15–20% of adults younger than 60 years with de novo AML (2, 3). The function of the genes involved in these translocations (4 –7) is modified as result of the fusion with inappropriate partners and results in disruption of their role in promoting transcriptional activity, by recruiting an active histone deacetylase (HDAC; Ref. 8) through interactions with the nuclear corepressors NCoR and Sin3A (9 –11). Quite recently, this molecular phenomenon has been recog- nized in acute promyelocytic leukemia (APL; PML/RAR and PLZF/ RAR positive) as well as CBF AML. This molecular alteration thus represents a shared pathogenetic mechanism (12). On this basis, efforts are directed to tailor derepressive, maturative therapy for specific AML subtypes by the use of histone deactylase inhibitor (HDACi) such as suberoylanilide hydroxamic acid (SAHA), trichos- tatin A (TSA), valproic acid (13, 14), and several others under investigation. Butyrate has long been known to be a HDACi able to induce maturation in normal and tumor cells and to have a partial activity in vivo in the therapy of myelodysplastic syndromes, refractory AML and APL (15–17). Recently, monosaccharide ester derivatives of butyric acid characterized by high molecular stability have been developed and studied (18). We demonstrated previously that sodium butyrate and monosaccharide esters inhibit cell growth and induce incomplete maturation and apoptosis in AML cells (19). We provide here evidence for the acetylating activity of sodium butyrate and a xylitol butyrate derivative (D1) in two AML cell lines and in primary AML blasts. Histone acetylation was paralleled by terminal granulo- cytic maturation, apoptosis, and inhibition of proliferation in the CBF AML cell line Kasumi-1 and in the CBF AML primary blasts, whereas in other non-CBF AML cases, maturation was incomplete or absent. MATERIALS AND METHODS Cells and Culture Conditions. Heparinized peripheral blood and bone marrow samples were obtained after informed consent from 19 patients with AML. French-American-British diagnosis included 5 M1, 6 M2, and 8 M4 (20). T lymphocytes were removed after Ficoll-Isopaque centrifugation by E-rosetting with sheep RBCs and AML cells, which were then cryopreserved in DMSO, as described previously (21). After being thawed and washed, AML blasts were depleted of adherent cells after incubation in serum-free medium in plastic culture flasks (250 ml; Greiner) and then cultured. After this procedure, blast content reached 98 –100% in each sample, as revealed by morphological analysis. Cytogenetic analysis and reverse transcription-PCR to establish the presence of AML-associated fusion genes were performed according to stand- ard methods (22). Primary AML blasts, Kasumi-1 cells (a human AML1/ETO- positive cell line derived from a myeloblastic leukemia; Ref. 23), and the human pre-osteoclastic leukemic cell line FLG 29.1, derived from a mono- blastic leukemia (kindly provided by Dr. Bernabei, AOUC Careggi, Firenze, Italy; Ref. 24), were cultured in RPMI 1640 supplemented with 50 units/ml penicillin, 50 g of streptomycin, and 10% FCS at 37°C in a humidified atmosphere containing 5% CO 2 . Kasumi-1 cells (0.4  10 6 /ml) and AML blasts (1  10 6 /ml) were incubated in the presence of different doses of sodium butyrate (Sigma) or D1 (O-n-butanoil-2,3-O-isopropylidene--D-manno- furanoside; kindly provided by Chiesi Pharmaceuticals, Varese, Italy). Cell Proliferation and Maturation. Cell proliferation was evaluated and quantified by cell counting. Differentiation was evaluated by morphological, cytochemical, and immunological examinations. Before and after treatment of culture, cytospin preparations of AML cells were stained with May-Gru ¨nwald/ Giemsa. Morphology of cells was then examined by light microscopy using a 100 lens and immersion oil. At least 200 cells/slide were counted in dupli- cate, and the percentages of blasts, promyelocytes, myelocytes, metamyelo- cytes, band forms, granulocytes, monoblasts, monocytes, and macrophages were scored. Maturation morphological signs were considered to be appear- ance of cytoplasmic granules, loss of cytoplasmic basophilia, chromatin con- densation, and nuclei segmentation. Nucleated cell number and viability were determined by counting with Tu ¨rck solution and trypan blue dye exclusion. Picnosis of nuclei and cytoplasmic condensation were also scored as specific Received 3/13/03; revised 10/8/03; accepted 10/15/03. Grant support: Associazione Italiana per la Ricerca sul Cancro, Associazione Italiana per la lotta contro le Leucemie, Ministero per la Istruzione, l’Universita ` e la Ricerca, and Ente Cassa di Risparmio di Firenze. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Notes: A. G. was supported by Associazione Italiana contro le Leucemie. E. R. was the recipient of a fellowship from Fondazione Italiana per la Ricerca sul Cancro. Requests for reprints: Valeria Santini, Divisione di Ematologia, Universita ` degli Studi di Firenze, Policlinico di Careggi, Viale GB Morgagni 85, 50134 Firenze, Italy. Phone: 390554277296; Fax: 39055412098; E-mail: santini@unifi.it. 8955 Research. on November 9, 2015. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from