NFKB1 Is a Direct Target of the TAL1 Oncoprotein in Human T Leukemia Cells Pei-Yun Chang, 1 Kyle Draheim, 2 Michelle A. Kelliher, 2 and Shigeki Miyamoto 1 1 Program in Molecular and Cellular Pharmacology, Department of Pharmacology, University of Wisconsin-Madison, Madison, Wisconsin and 2 Department of Cancer Biology and the Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts Abstract We recently showed that a subset of human T acute lymphoblastic leukemia (T-ALL) cell lines expresses low basal levels of p50, a nuclear factor-KB (NF-KB)/Rel family member, resulting in their capacity to activate the atypical p65:cRel complex rather than the classic p50:p65 dimer. Here, we show that the transcription factor TAL1 (also known as SCL) binds to the promoter of the NFKB1 gene that encodes p50 and represses its transcription to set up this unique response in T-ALL cells. When TAL1 expression is reduced in CEM T leukemia cells, basal NFKB1 expression is increased, and the levels of p65:cRel complex and transcrip- tion of its target gene, such as intercellular adhesion molecule-1 (ICAM-1), are reduced in response to etoposide treatment. Moreover, a significant negative correlation between NFKB1 and TAL1 or LMO1 was found in primary human TAL1/LMO1 double-positive T-ALL samples previously described by Ferrando et al. Thus, TAL1 modulates NFKB1 expression and an NF-KB-dependent transcriptional program in a subset of human T-cell leukemia cells. (Cancer Res 2006; 66(12): 6008-13) Introduction T-cell acute lymphocytic leukemia 1 (TAL1, also known as the stem cell factor SCL) is a member of the basic helix-loop-helix family of transcription factors and is normally involved in regulation of hematopoiesis (1). In the T-cell lineage, TAL1 is active only at early thymocyte stages and regulates expression of pre-Ta , Rag2, and cyclin D1 (2–4). Aberrant expression of TAL1 in later stages of T-cell development is associated with T-ALL (5). We have recently described that certain T-ALL cell lines express low basal levels of p50, a member of the nuclear factor- nB (NF-nB)/Rel family of transcription factors (6). This is associated with the formation of an atypical p65:cRel heterodimer, instead of the canonical p50:p65 heterodimer, and activation of p65:cRel target genes, such as the intercellular adhesion molecule-1 (ICAM-1) gene (7). When these cells are exposed to repetitive NF-nB activation stimuli, an augmented NF-nB response can be observed, leading to an enhanced cell survival response against an anticancer agent etoposide (6). Below, we describe evidence that NFKB1, which encodes the p50 protein, is a novel target of TAL1 to set up this unique response in T-ALL cells. Materials and Methods Antibodies and chemicals. Immunoglobulin G (IgG) antibodies against actin (C-11), p65 (C-20), and RelB (C-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-cRel antibody (SA-172) was obtained from Biomol (Plymouth Meeting, PA). Anti-p52 (06-413) and anti-p50 (06- 886) antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). Horseradish peroxidase–conjugated protein A and horseradish peroxidase–conjugated anti-rabbit and anti-mouse antibodies were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Etoposide (VP16) was purchased from Sigma-Aldrich (St. Louis, MO). General protocols. Electrophoretic mobility shift assays (EMSA; including the Ign-nB and p21-nB probe oligonucleotide probe) were done as previously described (6). The ICAM1-nB probe is 5¶-TTAGCTTG- GAAATTCCGGAGCGAGAGGCCAT-3¶. Immunoprecipitation and Western blotting were done as described previously (6). All experiments were repeated at least thrice unless otherwise specified, and the results were quantified by exposing dried EMSA gels on a phosphoimager screen and analyzed by the IQMac1 program. Chromatin immunoprecipitation analysis. Chromatin immunopre- cipitation solutions were purchased from Upstate Cell Signaling (Charlottes- ville, VA), and the assays were done according to the manufacturer’s protocol with the minor modifications (as described in ref. 6). In all chromatin immunoprecipitation experiments, quantitative real-time data are presented by setting the untreated serum precipitated samples as unity. The average and SDs were calculated by the Microsoft Excel program and plotted by the KaleidaGraph software. Forward-NFKB1-promoter primer 5¶- GAATTCCATGGATGGCAGAAGATGATCCATAT-3¶ and reverse-NFKB1-pro- moter primer 5¶-GAATTCCTAGCTCATCAATGCTTCATCCC-3¶. Quantitative reverse transcription-PCR analysis. Total RNA from various cell types was extracted with the Qiagen RNeasy kit. cDNA was synthesized by as previously described (6). Quantitative real-time reverse transcription-PCR (RT-PCR) reactions (25 AL) contained 2 AL of cDNA, 12.5 AL of SYBR Green (Applied Biosystems, Foster City, CA), and the appropriate primers. Product accumulation was monitored by SYBR Green fluorescence with ABI Prism 7000 Sequence Detection Systems. The relative expression levels were determined from a standard curve of serial dilutions of cDNA samples. Forward and reverse primers for real-time RT-PCR are previously described (5, 6). The average and SDs were calculated by the Microsoft Excel program and plotted by the Kaleida- Graph software. Generation of stable AS- TAL1 CEM cells. The human TAL1 cDNA was cloned in the antisense orientation by digesting the plasmid pMSCL (8) with Eco RI and religation into the pcDNA3 vector. The Eco RI sites come from the plasmid polylinker. The integrity of the expression vector was confirmed by direct sequencing. These constructs were then electroporated into CEM cells and selected with G418 (1 mg/mL). Isolation of thymocytes and thymomas. Generation of transgenic animals were described previously (9). Tumors were isolated from leukemic transgenic mice and converted into cell culture. Thymi were isolated from 4-week-old mice. For protein extraction, cells were lysed in NP40 lysis buffer [150 mmol/L NaCl, 1% NP40, 50 mmol/L Tris (pH 8)]. Whole RNA extracts were prepared using TRIzol reagent (Invitrogen, San Diego, CA). Statistical analysis. The statistical analysis was done by Graph Pad Prism program (t test, polynomial, or logarithmic regression) and plotted with Microsoft Excel program. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Shigeki Miyamoto, Department of Pharmacology, University of Wisconsin, 301 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706. Phone: 608-262-9281; Fax: 608-262-1257; E-mail: smiyamot@ wisc.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-0194 Cancer Res 2006; 66: (12). June 15, 2006 6008 www.aacrjournals.org Priority Report