ABSTRACT: Purpose: Treatment of acute promyelocytic leukemia (APL) with the retinoid, all trans retinoic acid (ATRA), along with standard chemotherapy has significantly improved survival compared to chemotherapy alone1. ATRA mediates its benefit in APL by overcoming the transcriptional block mediated by PML-RARï¡ fusion oncoprotein, thereby, restoring expression of retinoid response genes2. The therapeutic benefits observed with ATRA treatment in APL have not been achieved in the other more common sub-types of acute myeloblastic leukemia (AML)3. This likely reflects the recruitment of histone deacetylase complexes by other recurrent chromosomal translocations in AML cells4. In this experiment, we evaluated if treatment of the AML cell line, OCI/AML-2, with the histone deacetylase inhibitor, valproic acid (VPA), would alter the expression of sub-sets of genes by itself or in conjunction with ATRA that have been shown to be modulated by ATRA in APL2. Experiment Overall Design: Methods: OCI/AML-2 cells were cultured at 37°C and 5% CO2 in ï¡MEM growth media supplemented with antibiotics and 10% fetal calf serum (HyClone, UT, USA). The drugs, ATRA and VPA, were obtained from Sigma-Aldrich Canada Ltd. (ON, CAN) and were re-suspended in 100% ethanol. OCI/AML-2 cells were treated with ATRA as a single 10-6 M dose and VPA was dosed at 0.6mM every 8 hours over 24 hours. The following four treatment combinations were evaluated: i) controls (solvent only) ii) ATRA iii) VPA iv) ATRA + VPA. Experiment Overall Design: Suspension cells were pelleted at 300 g 24 h after initial drug exposure. Total RNA was isolated using an RNeasy kit (Qiagen Inc., Canada) and stored at â70°C in DEPC-H2O. Total RNA was then further concentrated to 2.5µg/ïL by ethanol precipitation overnight at â20°C in 0.3M sodium acetate pH 5.2 (Sigma-Aldrich Canada Ltd.). Experiment Overall Design: GeneChip experiments were carried out by the Microarray Facility, The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON. A total of 20µg of total RNA was used to generate biotin labeled cRNA probes according to Affymetrixâs recommended protocol using a labeling kit from EnzoBiochem, Inc. (NY, USA). A total of 15µg of the biotin labeled cRNA was hybridized onto the human U133A Affymetrix GeneChip. The GeneChip was washed as per the Affymetrix EukGE-ws2v4 protocol available at (https://www.affymetrix.com/site/login/login.affx). The GeneChip was scanned using a GeneArray 2500 scanner (Agilent, CA, USA) and hybridization intensity was obtained and detection p value and signal were calculated using MAS 5.0 software. Target signal was set to 150 for scaling for all of the experiments. The output chp file was exported to text file and used for further analysis with the Multiple experiment viewer (MeV) version 2.2 available from the Institute for Genomic Research at http://www.tigr.org/. Signal intensities obtained from the OCI/AML-2 cells treated with ATRA, VPA, or ATRA + VPA were compared to untreated OCI/AML-2 controls. Only genes that were identified as present (P) and displayed more than two fold change in signal intensity compared to untreated cells (controls) were carried forward for further analysis. Gene expression experiments were normalized using total intensity, median centered and log(2) transformed to give equal weight to expression values relative to the median for analysis. Unsupervised hierarchical clustering was performed using average linkage and Pearson correlation as a measure of distance. Experiment Overall Design: Results: Relatively few genes were regulated by ATRA treatment in OCI/AML-2 cells. However, treatment of OCI/AML-2 cells with VPA altered the expression of genes that encode for proteins involved with regulation of cell cycle, interferon response, apoptosis and differentiation. Examples of genes depressed by VPA, but not ATRA, involved regulators of cell cycle, cyclin A2 and MYC. The combination of ATRA + VPA further suppressed expression of these genes. Genes such as IRF1, neutrophilic cytosolic factor, ICAM3 and C/EBPï¢ increased in response to ATRA in OCI/AML-2 cells whereas VPA did not alter their expression over untreated OCI/AML-2 cells. However the combination of ATRA + VPA further enhanced the expression of these genes. Examples of genes induced by VPA that were not affected by ATRA in OCI/AML-2 cells include cell surface markers CD9, and MHC class II, promoters/enhancers of apoptosis such as MAPK kinase, interferon responsive genes, and caspase 7, and negative regulators of cell cycle like p21 and p19. The addition of ATRA to VPA increased the expression of some, but not all of these genes. It would appear that VPA dramatically affects the gene expression pattern of OCI/AML-2 cells, and modulates the expression of a set of genes, similar to those modulated by ATRA in APL cells2. Experiment Overall Design: References Experiment Overall Design: 1. Tallman MS, Andersen JW, Schiffer CA, Appelbaum FR, Feusner JH, Ogden A, Shepherd L, Experiment Overall Design: Willman C, Bloomfield CD, Rowe JM, Wiernik PH. All-trans-retinoic acid in acute Experiment Overall Design: promyelocytic leukemia. N Engl J Med 1997; 337(15):1021-1028. Experiment Overall Design: 2. Tamayo P, Slonim D, Mesirov J, Zhu Q, Kitareewan S, Dmitrovsky E, Lander ES, Experiment Overall Design: Golub TR. Interpreting patterns of gene expression with self-organizing maps: methods Experiment Overall Design: and application to hematopoietic differentiation. Proc Natl Acad Sci U S A 1999; Experiment Overall Design: 96(6):2907-2912. Experiment Overall Design: 3. Estey EH, Thall PF, Pierce S, Cortes J, Beran M, Kantarjian H, Keating MJ, Andreeff Experiment Overall Design: M, Freireich E. Randomized phase II study of fludarabine + cytosine arabinoside + Experiment Overall Design: idarubicin +/- all-trans retinoic acid +/- granulocyte colony- stimulating factor in poor Experiment Overall Design: prognosis newly diagnosed acute myeloid leukemia and myelodysplastic syndrome. Experiment Overall Design: Blood 1999; 93(8):2478-2484 Experiment Overall Design: 4. Gelmetti V, Zhang J, Fanelli M, Minucci S, Pelicci PG, Lazar MA. Aberrant Experiment Overall Design: recruitment of the nuclear receptor corepressor-histone deacetylase complex by the acute Experiment Overall Design: myeloid leukemia fusion partner ETO. Mol Cell Biol 1998; 18(12):7185-7191.
