Induction of Apoptosis and Cell Cycle Arrest in Acute Myeloid Leukemia Cells by Histone Deacetylase Inhibitor, Suberoyl Hydroxamic Acid (SAHA): Lack of Cross-Resistance to Cytosine Arabinoside and Etoposide.

Abstract

Inhibitors of histone deacetylases (HDAC) such as SAHA are being introduced into the clinical treatment of hematopoietic neoplasms. These compounds can induce apoptosis or cell cycle arrest by modification of the chromatin structure of malignant cells, thereby modulating the expression of target genes. We investigated the effects of HDAC inhibitors, SAHA and trichostatin A, in twelve acute myeloid leukemia (AML) cell lines. Cytotoxicity was determined with the use of a tetrazolium-based colorimetric assay. The 50% inhibition concentrations (IC50) of SAHA were in the micromolar, of trichostatin A in the nanomolar range. Three cell lines were 3–5 times more resistant to both HDAC-inhibitors than the other leukemias, namely the myelomonocytic leukemia ML-2, and the erythroleukemias HEL and K562. All investigated AML cells were, however, more sensitive to SAHA than three patient samples of normal CD34+ progenitor cells mobilized into peripheral blood. A close association between the IC50 of TSA and SAHA was noted within the panel of AML lines (r=0.78). In contrast, chemosensitivity to HDAC-inhibitors was not significantly correlated with IC50 for etoposide, cytosine arabinoside, or staurosporine, respectively. To distinguish between growth arrest and induction of apoptosis by SAHA, we analyzed the cell cycle status by staining with propidium iodide, and exposure of phosphatidylserine by an Annexin V assay. Cell cycle arrest in G1 phase was observed in four AML cell lines with an increase of G1 cells by 20–49% in comparison with untreated cells. One cell line, KG-1a, displayed G2 arrest. SAHA induced apoptosis in eight cell lines with one line displaying both apoptosis and G1 cell cycle arrest simultaneously. KASUMI-1 cells bearing the AML1/ETO gene fusion, a target for HDAC's, underwent apoptosis upon exposure to SAHA. Constitutive expression of the cell cycle inhibitor p27Kip1, determined by Western blotting, was associated with increased numbers of G1 cells following treatment with SAHA (p=0.036, one-tailed Mann-Whitney test). Conversely, constitutive expression of cyclins A, B1, D3 and E, as well as p53 and p16INK4 was not predictive for induction of cell cycle arrest. Expression of P-glycoprotein (ABCB1) as assessed by flow cytometry was not correlated with the IC50 for SAHA. The anti-apoptotic proteins Bcl-2, Mcl-1 and XIAP were analyzed by Western blotting. No association with resistance to SAHA-induced apoptosis was noticable. In summary, AML cells from permanent lines were more sensitive to SAHA than normal hematopoietic progenitor cells. The potential role of p27Kip1 in modulation of the cytotoxicity of HDAC inhibitors requires further study. Lack of cross-resistance with drugs used in clinical treatment suggests that HDAC inhibitors may be useful for treatment of chemoresistant AML.