1563-1569 7/11/07 19:04 Page 1563 ONCOLOGY REPORTS 18: 1563-1569, 2007 Induction of apoptosis increases expression of non-canonical WNT genes in myeloid leukemia cell lines HAKKI OGUN SERCAN1, MELEK PEHLIVAN1, OZLENEN SIMSEK1, HALIL ATES2 and ZEYNEP SERCAN1 Depatments of 1Medical Biology and Genetics, 2Hematology/Oncology, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey Received May 3, 2007; Accepted July 20, 2007 Abstract. With the aim of determining the differential and cyclin D. The presence or exclusion of co-receptors (low expression of WNT and FZD genes, before and after density lipoprotein receptor-related protein 5 and 6-LRP5/6) induction of apoptosis in BCR-ABL positive cells, we treated or secreted Wnt antagonists (soluble frizzled related proteins the myeloid cell line K562 and control cell line HL60 with - sFRPs- and Dickkopf proteins) increases the complexity of imatinib mesylate and etoposide, and analyzed relative the molecular mechanisms underlying cellular outcomes. mRNA expression levels of WNT, FZD and sFRP genes Non-canonical pathway activation is independent of ß- under normal and apoptotic conditions by real-time RT-PCR. catenin. Two non-canonical pathways have also been defined: We observed marked increase in mRNA levels of FZD4, the planar cell polarity (PCP) and the Wnt/Ca2+ pathways (3-6). FZD5, FZD7 and WNT5b, correlating with apoptotic activity There is an overlap of proposed Wnt and Fzd proteins and and independent of the agent or cell line used. Our results secondary messengers participating in these non-canonical suggest the involvement of non-canonical Wnt signaling in pathways, complicating further the interpretation of experi- executing programmed cell death in myeloid cell lines. mental data on regulation, ligand-receptor partners, and inter- mediate members of non-canonical signaling. Although our Introduction understanding of non-canonical signaling, especially during adult homeostasis is limited; it has been proposed by different Wnt proteins are a large family of secreted, lipid modified, authors with supporting experimental data that non-canonical cystein-rich glycoproteins that serve as ligands for the signaling antagonizes the canonical/ß-catenin pathway by frizzled (Fzd) family of seven-transmembrane receptors (1). exerting signals with opposite outcomes (7,8). Wnt/Fzd signaling activates evolutionary conserved signaling Wnt molecules have been grouped as canonical and non- pathways that have crucial roles in development by regulating canonical pathway activators. However this classification is proliferation, differentiation, migration and cell-cell adhesion, not a clear-cut distinction. There are reports showing that a depending on the cellular context. The wide variety of pro- given Wnt protein may activate different signaling pathways cesses that Wnt signaling controls has made it a crucial depending on the cellular context, binding specificities to component of not only development but also adult homeostasis. frizzled receptors, experimental conditions and the presence, Ligand-receptor specificity between the identified 19 Wnt absence or pattern of co-receptors available (9-11). In short and 10 Fzd proteins is largely unknown with limited data on the ability of a given Wnt molecule to trigger canonical or appropriate ligand-receptor combinations activating distinct non-canonical signaling is not absolute (9). intracellular signaling pathways (1,2). Wnt signaling can be Deregulated Wnt signaling has been implied in a variety classified as canonical and non-canonical pathways, where the of human diseases including cancer (12). These studies are canonical pathway is responsible for preventing the degra- mainly limited to the canonical pathway where known proto- dation of ß-catenin. Wnt-frizzled receptor coupling results in oncogene and tumor suppressor gene products function as the stabilization and nuclear translocation of ß-catenin. intermediate signal transducers. There are numerous reports Accumulation of ß-catenin in the nucleus leads to the tran- linking ß-catenin stabilization with different forms of solid scriptional activation of multiple target genes such as c-myc tumors as well as leukemia. Aberrant Wnt signaling has been reported in a number of leukemia of both lymphoid and _________________________________________ myeloid lineages (13). Nature of leukemias differ from solid tumors in that the hematopoietic system of the adult organism Correspondence to: Dr Zeynep Sercan, Department of Medical is composed from cells generally with a short life span which Biology and Genetics, Dokuz Eylul University, Faculty of Medicine, are renewed by differentiating from a small population of Inciralti, 35340 Izmir, Turkey hematopoietic stem cells (HSCs). Many leukemias are either E-mail: [email protected] identified or associated with specific chromosomal trans- locations resulting in oncogenic fusion products that have Key words: Wnt proteins, frizzled receptor, apoptosis, bcr-abl, been shown to block differentiation and/or escape apoptosis imatinib and/or induce proliferation or expansion of the transformed cell population. There is growing evidence suggesting Wnt 1563-1569 7/11/07 19:04 Page 1564 1564 SERCAN et al: INCREASED EXPRESSION OF NON-CANONICAL WNT GENES signaling plays an important role in normal HSC renewal and genes. In treated cells, K562 and HL60 cells were seeded that dysregulation of this pathway may lead to leukemia (13). 1.5x106 cells/ml in serum-free, phenol red-free medium in Chronic myeloid leukemia (CML) is of interest because the 25 cm2 cell culture flasks. After 24 h, 5 μM imatinib or 30 μg/ initial transformation event occurs in an HSC. The ml etoposide was added to each flask and incubated for 6, 12 Philadelphia chromosome, der(22)t(9;22)(q34;q11), is the and 24 h. Cells were harvest and total RNA was extracted hallmark of CML resulting in the Bcr-Abl chimeric using RNeasy® Mini kit (Qiagen). To avoid genomic DNA oncogene. Reports on the role of Wnt signaling in HSC contamination, RNA samples were treated with RNase-free renewal, ß-catenin accumulation and canonical pathway DNase I (Qiagen) following manufacturer's instructions. activation in CML and the Bcr protein defined as a negative RNA integrity and purity were verified by electrophoresis regulator of Wnt signaling, together has provided a line of and OD260/OD280 nm absorption ratio respectively. cDNA evidence suggesting deregulation of the pathway may have a was synthesized by using SuperScript™ First Strand kit role in disease pathogenesis (13,14). BCR-ABL positive cells (Invitrogen), 2 μg of total RNA and random primers according are shown to be resistant to apoptosis, a feature thought to to the manufacturer's instructions. contribute to the expansion of the malignant cell clone (15). How Wnt signal transduction interacts with apoptotic signaling Real-time RT-PCR. Real-time PCR was carried out using a is a less explored area of research and interestingly there is Light Cycler® 2.0 (Roche Diagnostics) instrument and Light scarce evidence that Wnt proteins are directly involved in Cycler FastStart DNA MasterPLUS SYBR Green I (Roche programmed cell death, a crucial process of development. In Diagnostics) kit. Reactions were performed in a 20-μl volume light of the previously reported ß-catenin accumulation and with 5 pmol of each primer and 5 μl of cDNA template canonical pathway activation, along with reported resistance to derived from reverse transcribed RNA of untreated, 6, 12 and apoptosis of the malignant clone in CML; we aimed to 24 h treated cells. ß-actin gene was used as endogenous control determine the differential expression of genes involved in and reference gene for relative quantifications. Primer Wnt signaling, before and after induction of apoptosis in sequences are given in Table I. The same thermal profile BCR-ABL positive cells. was optimized for all primers: a pre-incubation for 10 min at 95˚C, followed by 40 amplification cycles of denaturation Materials and methods at 95˚C for 10 sec, primer annealing at 59˚C for 5 sec, and primer extension at 72˚C for 10 sec. H2O was included as a no Cell lines and chemicals. K652 is a BCR/ABL positive cell template control. Melting curves were derived after 40 cycles line derived from a CML patient in erythroid blast crisis. by a denaturation step at 95˚C for 10 sec, followed by annealing HL60 is a BCR/ABL negative cell line derived from an acute at 65˚C for 15 sec and a temperature rise to 95˚C with a promyeloblastic leukemia (APL) patient. HL60 was used as a heating rate of 0.1˚C per second and continuous fluorescence BCR/ABL negative myeloid leukemia cell line control in the measurement. Final cooling was performed at 40˚C for 30 sec. apoptotic induction experiments. Both cell lines were purchased Melting curve analyses of each sample were done using from DSMZ (Deutsche Sammlung von Mikroorganismen LightCycler Software version 4.0.0.23 (Roche Diagnostics). und Zellkulturen GmbH) and grown in RPMI-1640 medium The analysis step of relative quantification is a fully automated supplemented with 15% FBS and 1% L-glutamine; in a 5% process done by the software, with the efficiency set at 2 CO2 saturated incubator at 37˚C. and the cDNA of untreated cells defined as the calibrator. All Imatinib was kindly provided by Novartis Company (Basel, samples in glass capillaries were later run in 2% agarose Switzerland). K562 cells seeded at a 5x104 cells/well density electrophoresis to check for size and non-specific amplifi- in 96-well plates and were treated with imatinib at 2-, 5- and cations. All experiments were done in triplicates. 10-μM concentrations. Caspase 3/7 activation was evaluated by a caspase-3/7 luminometric assay (see below). No significant Measurement of caspase-3 activity. Caspase activation was difference of caspase activation was observed between 5- determined by detecting caspase-3 activity in cell lysates and 10-μM concentrations and 5 μM imanitib was used in using the Caspase-Glo 3/7 Assay Kit (Promega) following subsequent experiments where mRNA levels were analyzed.