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Sirna Targeting the IRF2 Transcription Factor Inhibits Leukaemic Cell Growth

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Sirna Targeting the IRF2 Transcription Factor Inhibits Leukaemic Cell Growth 175-183 9/6/08 16:54 Page 175 INTERNATIONAL JOURNAL OF ONCOLOGY 33: 175-183, 2008 175 siRNA targeting the IRF2 transcription factor inhibits leukaemic cell growth AILYN CHOO1, PATRICIA PALLADINETTI1, TIFFANY HOLMES2, SHREERUPA BASU2, SYLVIE SHEN1, RICHARD B. LOCK1, TRACEY A. O'BRIEN2, GEOFF SYMONDS1 and ALLA DOLNIKOV1,2 1Children's Cancer Institute Australia for Medical Research; 2Sydney Cord & Marrow Transplant Facility, Sydney Children's Hospital, High Street, Randwick NSW 2031, Australia Received January 11, 2008; Accepted February 27, 2008 Abstract. Interferon regulatory factor (IRF) 1 and its functional mechanisms or transcriptional regulators thereby directly antagonist IRF2 were originally discovered as transcription interfering with haematopoietic cell growth and differentiation factors that regulate the interferon-ß gene. Control of cell (1). growth has led to the definition of IRF1 as a tumour suppressor Mutations that create constitutively active Ras proteins gene and IRF2 as an oncogene. Clinically, approximately are among the most frequently detected genetic alterations in 70% of cases of acute myeloid leukaemia demonstrate dys- human leukaemia (2), occurring in approximately 30% of regulated expression of IRF1 and/or IRF2. Our previous AML cases (3). While frequently activated in haematopoietic studies have shown that human leukaemic TF-1 cells exhibit malignancies, the manner in which Ras activation contributes abnormally high expression of both IRF1 and IRF2, the latter to human leukaemia is not well understood. The Ras protein acting to abrogate IRF1 tumour suppression, making these has a crucial role in many haematopoietic regulatory processes cells ideal for analysis of down-regulation of IRF2 expression. and has been the target of many therapeutic approaches (3,4). A novel G418 screening protocol was developed and used for Mutant N-ras (N-rasm) is known to impair murine myelopoiesis identifying effective siRNA that targets IRF2 (siIRF2). Using and erythropoiesis both in ex vivo tissue culture models and optimized siIRF2 in leukaemic TF-1 cells, IRF2 was down- in vivo in bone marrow reconstituted mice where it confers regulated by approximately 70% at both mRNA and protein susceptibility to haematopoietic disorders that resemble human levels. Phenotypically, this resulted in growth inhibition AML (4). In addition, N-rasm was shown to induce myelo- associated with G2/M arrest as well as induction of polyploidy, proliferation when transduced into human primitive HSC/HPC differentiation and apoptosis. In contrast to these results, siIRF2 both ex vivo in long-term culture and in vivo in a ‘humanized' targeting did not affect normal haematopoietic stem/progenitor NOD/SCID mouse model (5). cell growth. These results indicate the potential utility of IRF2 Paradoxically, ectopic expression of N-rasm was shown to inhibition as a therapeutic approach to cancer. induce tumour suppressive pathways in some leukaemic cell lines, namely K562 and U937 but not in TF-1 cells (6). Introduction High-throughput microarray expression analysis revealed up-regulation of the transcription factor and tumour suppressor, Acute myeloid leukaemia (AML) can be viewed as a clonal interferon regulatory factor (IRF) 1 when N-rasm was consti- myeloproliferative disorder of the haematopoietic stem/pro- tutively expressed in leukaemic K562 and U937 cells; but not genitor cell (HSC/HPC) characterised by the rapid expansion in TF-1 cells that carries a mutation in the endogenous N-ras of differentiation-arrested cells of varying maturity, often gene and exhibited high basal levels of IRF1 (6). Increased caused by distinct molecular mechanisms (1). These molecular levels of IRF1 in K562 and U937 cells induced up-regulation mechanisms include leukaemia-associated fusion proteins and of the tumour suppressor gene, p21WAF1 and as expected, mutations that function to aberrantly activate either signalling antisense-mediated IRF1 suppression resulted in reduced levels of p21WAF1 (6). Results indicated that this activation cascade is responsible for the tumour suppressor effects _________________________________________ observed in K562 and U937 cells (6). Up-regulation of IRF1 via ectopic expression of N-rasm may provide a potential tumour suppressive approach for leukaemia therapy (6). Correspondence to: Dr Alla Dolnikov, Sydney Cord & Marrow The IRF1 gene is located on chromosome 5q31.1, an area Transplant Facility, High Street, Randwick NSW 2031, Australia commonly deleted in myelodysplasia, AML and acute pro- E-mail: [email protected] myelocytic leukaemia (APL) resulting in inactivation of one or both alleles (7-9). Key words: interferon regulatory factor 1 and 2, leukaemogenesis Surprisingly, in a recent study, IRF1 was found to be highly expressed in a significant proportion of primary AML specimens (10). In addition, high IRF1 expression was seen 175-183 9/6/08 16:54 Page 176 176 CHOO et al: siRNA TARGETING THE IRF2 TRANSCRIPTION FACTOR INHIBITS LEUKAEMIC CELL GROWTH in human leukaemia TF-1 cells (6). We hypothesized that fection Kit Protocol (Qiagen Inc.). All experiments were IRF1 tumour suppressor activity was abrogated in these performed in triplicate and compared to cells incubated in AMLs by secondary mutations (11). Retroviral cDNA library lipofection reagent alone (untreated). For electroporation analysis was performed to screen for the genes that abrogate experiments, 1x107 TF-1 cells were resuspended in 500 μl N-rasm-induced growth arrest (11). This analysis identified serum-free RPMI media containing 3 μM siRNA. Cells were IRF2 as an abrogating factor in the growth arrest pathway electroporated at 250 V, 500 uF. siRNA uptake was analysed (11). IRF2 over-expression in U937 cells abrogated N-rasm- in the FITC-labelled scrambled oligonucleotide control at 6 h induced growth suppression through the repression of p21WAF1 for both lipofection and electroporation. siRNA uptake was (11). In addition, high IRF2 expression was found in leukaemia analysed by flow cytometry. TF-1 cells (6). Overall, these results established an N-rasm mediated growth suppression pathway whereby the balance Flow cytometry. Flow cytometry analyses were performed between IRF1 and IRF2 expression (IRF1:IRF2 ratio) appears using a FACSCalibur instrument and CellQuest analysis to be the determining factor. software (Becton Dickinson, San Jose, CA). Events (1x104) We report here that down-regulation of IRF2 by specific were collected in each experiment. The FL1 channel was siRNA in TF-1 cells increases the IRF1:IRF2 ratio allowing used to measure FITC-labelled siRNA and siRNA uptake was IRF1 and p21WAF1-related tumour suppressor effects. These measured in live cells; gating based on forward (FSC) and effects included growth suppression, cell cycle arrest and side scatter (SSC) characteristics. The percentage of FITC- concomitant polyploidisation. In addition, targeting IRF2 in labelled cells and mean fluorescent intensity (MFI) were leukaemic TF-1 cells resulted in a reduced fraction of clono- analysed in each sample. For phenotyping, cells (1x106) were genic leukaemic blasts. By contrast, IRF2 down-regulation in blocked with mouse IgG and stained with monoclonal normal cord blood CD34+ primitive progenitor cells did not antibodies against CD45, CD34, CD33, CD14 and CXCR4 affect cell growth. To the best of our knowledge, IRF2 (Becton Dickinson). Events (1x105) were acquired and data targeting is a novel concept that may have therapeutic potential. were analysed with CellQuest (Becton Dickinson). CD34+ percentages were calculated as percentages of the lymphocyte Materials and methods gate (set on CD45+, low side scatter cells of the unexpanded UCB), excluding CD14+ cells. Propidium iodide (PI) was Cell culture. TF-1 cells were derived from the myeloid pro- included to assess UCB viability. genitor cell population recovered from a patient with erythro- leukaemia (American Type Culture Collection (ATCC), Clonogenic assays. Clonogenic assays were performed in Manassas, VA). U937 cells (ATCC) were derived from a Methylcellulose (StemCell Technologies Inc., Vancouver, patient's diffuse histiocytic lymphoma. Both cell types were BC, Canada). TF-1 cells (5x102) were cultured in 1 ml methyl- cultured in Roswell Park Memorial Institute (RPMI)-1640 cellulose in a 12-well plate for 8 days and single colony counts (Gibco BRL) medium containing 10% fetal calf serum were scored. Samples were then washed in PBS and counted (FCS), 2 mM L-glutamine, 50 U/ml penicillin, 50 μg/ml for statistical analysis. UCB CD34+ cells were plated in streptomycin, 25 mM HEPES buffer (RPMI medium) and Methocult GF H4534 [1% methylcellulose in IMDM, 30% -4 incubated at 5% CO2 and 37˚C. Recombinant interleukin-3 FBS, 1% BSA, 10 M 2-mercaptoethanol, 2 mM L-glutamine, (IL-3) (10 ng/ml) was also added for TF-1 cells. U937-derived 50 ng/ml SCF, 10 ng/ml granulocyte-macrophage colony- IRF2/Neo or Neo-only cells were produced as previously stimulating factor (GM-CSF), 10 ng/ml IL-3 (StemCell described (11). G418 antibiotic selection of these cells was Technologies)] supplemented with 3 U/ml of erythropoietin performed at 1000 μg/ml. Umbilical cord blood (UCB) was (EPO) and 50 ng/ml granulocyte-growth stimulating factor obtained from healthy full-term deliveries through the Australian (G-CSF) (R&D Systems) as previously described (5). Cells Cord Blood Bank, with Human Ethics approval and donor's (1x103) suspended in 1.1 ml were plated in each 35-mm dish. informed consent. UCB CD34+ cells
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