ª Oncogene (2002) 21, 5716 – 5724 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi) Monika Wilda1,2, Uta Fuchs1,2, Wilhelm Wössmann1,2 and Arndt Borkhardt*,1 1 Department of Pediatric Hematology and Oncology, University of Giessen, 35392 Giessen, Germany Short 21-mer double-stranded RNA (dsRNA) molecules have recently been employed for the sequence-specific silencing of endogenous human genes. This mechanism, called RNA interference (RNAi), is extremely potent and requires only a few dsRNA molecules per cell to silence homologous gene mRNA expression. We used dsRNA targeting the M-BCR/ABL fusion site to kill leukemic cells with such a rearrangement. Transfection of dsRNA specific for the M-BCR/ABL fusion mRNA into K562 cells depleted the corresponding mRNA and the M-BCR/ ABL oncoprotein. This was demonstrated by real-time quantitative PCR and Western blots. The BCR/ABL knockdown was accompanied by strong induction of apoptotic cell death. Leukemic cells without BCR/ABL rearrangement were not killed by M-BCR/ABL-dsRNA. In addition, to corroborate the extraordinary sequence specificity of RNAi, we designed another RNA oligo matching the M-BCR/ABL fusion site but having two point mutations within its central region. We show that these two point mutations abolished both p210 reduction and induction of apoptosis in K562 cells. Finally, we compared leukemic cell killing by RNAi to that caused by the ABL kinase tyrosine inhibitor, STI 571, Imatinib. For full induction of apoptosis, dsRNA targeting M-BCR/ ABL required 24 h more than Imatinib. This may be caused by the relatively long half-life of the BCR/ABL oncoprotein, which is not targeted by the RNAi mechanism, but is affected by STI 571. When we applied ds M-BCR/ABL RNA and STI 571 in combination, we did not observe a further increase in the induction of apoptosis. Nevertheless, these data may open a field for further studies towards gene-therapeutic approaches using RNA interference to kill tumor cells with specific genetic abnormalities. Oncogene (2002) 21, 5716 – 5724. doi:10.1038/sj.onc. 1205653 Keywords: RNA interference; BCR/ABL rearrangement; apoptosis; STI 571; gene therapy *Correspondence: A Borkhardt, Pediatric Hematology and Oncology, Feulgenstr.12, 35392 Giessen, Germany; E-mail: Arndt.Borkhardt@paediat.med.uni-giessen.de 2 These authors contributed equally to this work Received 9 March 2002; revised 9 May 2002; accepted 10 May 2002 Introduction Chromosomal translocations are the hallmark of many leukemias, lymphomas, and to a somewhat lesser extent, solid tumors. They frequently lead to the generation of chimeric fusion oncoproteins that trigger malignant transformation. Gene targeting of the chimeric fusion is an ideal way to kill the tumor cells specifically, while leaving the normal cells unaffected. In this light, antisense approaches using singlestranded molecules of either RNA or DNA are relatively straightforward techniques for these purposes. However, these attempts have consistently suffered from some profound problems, e.g. questionable specificity or rather low efficacy (Stein and Narayanan, 1994; Skorski et al., 1994; Smetsers et al., 1994). It has recently been discovered that short dsRNA molecules (siRNAs) trigger a complex mechanism, called RNA interference, that results in specific gene silencing even in human cells (Elbashir et al., 2001a). siRNAs are approximately 21 nucleotides in length and have a base-paired structure with 2nucleotide 3’-overhang. Chemically synthesized siRNAs may become powerful reagents for genomewide analysis of mammalian gene function in cultured somatic cells. Beyond their value for target validation, siRNAs also hold great potential as gene-specific therapeutic agents. The remarkable potency of the RNAi reaction enables a complete ‘knock-down’ of a specific protein and may therefore overcome some of the initial problems with conventional antisense strategies. Herein, we explored the feasibility of the RNAi reaction against an oncogenic fusion gene by targeting the M-BCR/ABL mRNA in K562 cells (Figure 1). The BCR/ABL rearrangement is the molecular equivalent of the Philadelphia chromosome, which arises from a reciprocal translocation between chromosomes 9 and 22 (Rowley, 1973). The translocation fuses two unrelated genes, BCR from chromosome 22 and ABL from chromosome 9, to form an oncogenic hybrid gene. With regard to the BCR gene, there are two breakpoint sites, M-BCR versus m-BCR, and hence two slightly different BCR/ ABL oncoproteins of 210 kD or 190 kD, respectively, are produced (Chan et al., 1987; Rubin et al., 1988; Hooberman et al., 1989). The resulting BCR/ABL protein has, as compared to the normal ABL protein, an increased kinase activity leading to pathological Killing of leukemic cells by RNA M Wilda et al 5717 Figure 1 (a) Schematic representations of the BCR/ABL hybrid gene. The gray colored zones within ABL, or BCR/ABL represent the area in which the taqman primers/probe are located. The region of the BCR/ABL fusion that was targeted by the dsRNA is indicated (upper case letters, BCR part; lower case letters ABL part). (b) Schematic structure of the dsRNA that cleaves the target mRNA. Sense and antisense sequence of both RNA strands that were annealed to dsRNA are shown. To control the specificity of the RNAi reaction, we additionally designed a siRNA oligo in which two base-pairs were mutated (lower case letters in italics, underlined). These two point mutations do not match within the sequence around the M-BCR/ABL fusion site. (c) Control of transfection efficiency by a FITC-labeled dsRNA. The siRNA was 5’-labeled with FITC and used for transfection. FITC-positive cells were visualized by fluorescence microscopy and calculated after counter-staining with DAPI phosphorylation of several downstream targets (Lugo et al., 1990; Goldman and Druker, 2001a). This results in oncogenic growth and inhibition of apoptosis (Druker et al., 1996). The expression of the BCR/ABL oncoprotein induces a disease resembling CML in mice (Daley et al., 1990). Cytogenetic and molecular studies of clinical samples revealed that the rearrangement is found in almost all patients with chronic myeloid leukemia (CML) and in approximately 30% of adults with acute lymphoblastic leukemia (ALL) (Dobrovic et al., 1991; Maurer et al., 1991; Westbrook et al., 1992). The latter subgroup usually has a poor response to conventional chemotherapy protocols and thus carries a dismal prognosis (Lestingi and Hooberman, 1993). However, we decided to target this particular rearrangement not only because of its high frequency and paramount prognostic importance. The small-molecule drug STI 571, now also known as Imatinib, inhibits the deregulated protein kinase ABL in Ph+ patients. It has dramatically improved the therapy for BCR/ABLpositive leukemias. Administered orally once daily, Imatinib had significant anti-leukemic effects even in patients in whom conventional treatment had failed. This was accompanied by readily tolerable side effects (Druker et al., 2001b,c). Experimentally, STI 571 suppressed proliferation of BCR/ABL-expressing cells and triggered their apoptotic death by various mechanisms (Druker et al., 1996; Carroll et al., 1997). In this study, we compared the efficiency of cell killing by STI 571 to that of BCR/ABL dsRNA in cells with M-BCR/ABL rearrangement. Results Reduction of BCR/ABL mRNA expression by dsRNA molecules A prerequisite for the therapeutic application of siRNAs is that the targeted cells or tissue contain a functional RNAi mechanism to bind to siRNAs and mediate mRNA degradation. Original reports about the successful induction of RNAi in cells of human origin primarily dealt with HeLa cells (Elbashir et al., 2001a). In order to test the activity of RNAi in K562 cells, we used a reporter gene assay. Plasmids coding for firefly and sea-pansy luciferase are co-transfected together with targeting and control siRNAs, and the relative luminescence of target and control luciferases is measured. When thus tested for their ability to specifically silence luciferase reporters K562 cells, encouragingly, were responsive to siRNAs (Figure 2a). The same protocol was used to test various commercially available liposomal transfection reagents (TransMessenger and Superfect from Quiagen, Hilden, Germany; Lipofectamine 2000, DMRIE-C, and Oligofectamine from Invitrogen, Paisley, UK) for their specific ability to deliver plasmid and dsRNA into Oncogene Killing of leukemic cells by RNA M Wilda et al 5718 Figure 2 (a) K562 cells were co-transfected with plasmids and dsRNA as indicated at the bottom of each bar. Cells were subjected to dual luciferase assay 48 h post-transfection. The luciferase reporter gene regions from plasmids GL3 (firefly) and pRT-TK (renilla) are used according to Elbashir et al., 2001a. The dsRNA Luc targets the firefly luciferase only, while leaving the renilla luciferase unaffected. The ratios of firefly and renilla luciferase are shown. A control panel was transfected with a dsRNA directed at the human MYC gene (right bars). The average of four independent experiments is shown, error bars indicate standard deviation, **statistically significant. (b) Example of a representative Lamin amplification plot of the taqman PCR. The Y-axis shows the threshold above baseline whereas the X-axis shows the number of PCR cycles. Pink curve (A): untreated K562 cells, Yellow (B): After treatment of K562 cells with dsRNA targeting Lamin, a higher number of PCR cycles is required to pass the fixed threshold (black horizontal line). (C) Treatment with dsRNA targeting BCR/ABL decreases the number of cycles required to pass the threshold, which indicates that slightly more Lamin mRNA is present in the sample. Please note that these values must be corrected according to the expression data of the housekeeping gene ABL. (c) Absolute copy number of Lamin mRNA per 10 000 copies of ABL in cells treated with dsRNA targeting BCR/ABL, Lamin or cells without dsRNA treatment. After 48 h, the amount of Lamin mRNA clearly decreases in the cells transfected with dsRNA against Lamin. (d) Western blot analysis of the Lamin A/C. Note the slight effect of as RNA. The blot was stripped and re-probed to check for equal loading of total protein K562 cells. On the basis of our Luciferase assays, we decided to choose Oligofectamine-based transfections for the subsequent experiments. Next, we checked whether an endogenous protein can be downregulated in K562 cells. We targeted an abundant protein, Lamin, for which previous studies also have convincingly demonstrated that it can be silenced by dsRNA in HeLa cells. Using a dsRNA oligo whose sequence has been published (Elbashir et al., 2001a), we evaluated whether the knock-down of Lamin can be reproduced in K562 cells. In addition, we wanted to calculate the achieved reduction of Lamin mRNA by quantitative real-time RT – PCR. In order to normalize for different qualities of input RNA, we used the expression of the housekeeper ABL gene. The Lamin mRNA was reduced to 22% of the corresponding value for the non-transfected controls (Figure 2b,c). Oncogene The cleavage of Lamin mRNA was accompanied by significant reduction of Lamin A/C protein as shown by Western blotting 48 h after transfection (Figure 2d). Thus, in K562 cells gene silencing is possible for both exogenously introduced and endogenously expressed transcripts. We next targeted the M-BCR/ABL mRNA and assayed its expression by taqman PCR. The BCR/ABL mRNA molecules were quantified and normalized to 10 000 molecules of housekeeper mRNA. To ensure that our quantitative PCR assay is not prone to artifacts, we used the expression levels of various housekeeper genes, the PBGD, the HPRT, the GUS and the TBP gene (Figure 3a). In contrast to the quantification procedure for Lamin mRNA, the expression of normal ABL mRNA was unsuitable for a housekeeper mRNA. The reason is that the taqman Killing of leukemic cells by RNA M Wilda et al 5719 Figure 3 (a) Copies of BCR/ABL mRNA per 10 000 copies of housekeeper mRNA (TBP, PBGD, GUS and HPRT) 48 h posttransfection. The BCR/ABL mRNA is reduced regardless of which housekeeper was used for normalization of RNA input. (b) Western blot analysis, cells were transfected with RNA as indicated at the bottom of each lane. In cells transfected with M-BCR/ABL dsRNA, the p210 was barely visible but normal p145 ABL is not affected ABL primer/probes are located in the region that is present in, and will equally amplify from, the BCR/ ABL fusion gene. Thus, siRNAs transfected would affect M-BCR/ABL mRNA as well as the normal ABL control RNA, making comparisons very difficult. As was found for Lamin mRNA, transfection of siRNA targeting the M-BCR/ABL fusion site downregulates the BCR/ABL expression, but to a somewhat lesser Oncogene Killing of leukemic cells by RNA M Wilda et al 5720 extent. The exact copy numbers of M-BCR/ABL in the various experimental conditions are given in Figure 3a. In general, the reduction of BCR/ABL mRNA was observed regardless of whether PBGD, HPRT, GUS or TBP was used for normalization of input RNA. Knockdown of M-BCR/ABL protein by dsRNA Next, we examined whether the p210 BCR/ABL protein is silenced in K562 cells, which would correspond to the significant reduction of the MBCR/ABL mRNA after transfection of a 21-mer dsRNA. As expected, p210 was reduced to an almost undetectable level in Western blots, whereas neither the wild-type ABL protein nor the Vimentin was influenced by the dsRNA M-BCR/ABL. To ensure sequence specificity of the M-BCR/ABL dsRNA we designed a dsRNA oligo, the sequence of which did not perfectly match the M-BCR/ABL fusion site. Specifically, we mutated the stretch of four adenosines in the targeted region from AAAA to ggAA (see Figure 1). This mutated siRNA was unable to reduce the p210 level, indicating the extraordinary sequence specificity of RNAi (Figure 3b). We finally wanted to test RNAiapproach for the m-BCR/ABL fusion as well. Unfortunately, we failed with various attempts to efficiently transfect SD-1 cells that display this minor fusion transcript (data not shown). Induction of apoptosis by ds M-BCR/ABL RNA and STI 571 Previous studies revealed that downregulation of BCR/ ABL renders K562 cells susceptible to induction of apoptosis by chemotherapeutic agents (McGahon et al., 1994). We looked for the induction of apoptosis 48 h and 72 h after transfection. As summarized in Figure 4a, 48 h after transfection with ds M-BCR/ABL the rate of apoptosis in K562 cells was above that in the controls but did not reach the same level as in the STI 571-treated cells. Twenty-four hours later, however, the number of Histone-associated DNA fragments had become the same in K562 cells treated with 1 mM STI 571 as in cells transfected with ds MBCR/ABL. In contrast, single-stranded antisense MBCR/ABL did not induce apoptosis above the control level. Perhaps not surprisingly, in our assay we did not see an additive effect when STI 571 and dsRNA were combined. To provide further evidence that neither the dsRNA itself nor the transfection reagent induces apoptosis, we transfected a series of cell lines (HeLa, 293, and Su-DHL) that do not contain a M-BCR/ABL rearrangement. In none of these cells was apoptosis induced (data not shown). When K562 cells were treated with a dsRNA that spans the m-BCR/ABL fusion, we also did not detect apoptotic cell death (Figure 4a). Finally, K562 cells that were transfected with the M-BCR/ABL siRNA having the two point mutations also failed to show apoptosis. These ELISA data were thus in good accordance with the lack of p210 reduction. Morphologically, we saw membrane Oncogene vacuolization and destruction in 82 or 57% of the K562 cells that were treated with 1 M STI 571 or transfected with dsRNA M-BCR/ABL, respectively. Again, cells transfected with either single-stranded asRNA against M-BCR/ABL or ds-m-BCR/ABL showed no signs of apoptosis above the control level (Figure 4b). Discussion In patients with leukemia and translocation t(9;22) or the BCR/ABL rearrangement, the molecular-targeted tumor therapy has dramatically improved through the development of the small-molecule drug, STI 571 (Goldman and Druker, 2001a; Druker et al., 2001a). It has considerable advantages over conventional treatment modalities (interferon alpha), e.g. rapid and more frequent hematological and cytogenetic responses combined with fewer side effects. In recent studies, several authors reported the development of resistance to STI 571, e.g. by genomic amplification of BCR/ ABL, increased expression of BCR/ABL mRNA or point mutation in the ABL gene (Mahon et al., 2000; Gorre et al., 2001; Hochhaus et al., 2001; Barthe et al., 2001). Thus, it is currently a common belief that STI 571 alone cannot cure CML or Ph+ positive ALL, and that the development of additional therapeutic approaches would be of interest for those patients (Goldman and Melo, 2001b). Towards this end, we have shown here that the specific silencing of BCR/ ABL mRNA by dsRNA-induced RNA interference is nearly as effective as STI 571 in tumor-cell killing. This approach, however, does not affect the oncoprotein itself but rather its corresponding fusion mRNA. In the light of the relatively long half-life of the BCR/ABL protein (Dhut et al., 1990) it seems understandable that the cells were killed less rapidly than they are by STI 571. The cell killing seen in our experiments can clearly be attributed to an RNAi effect, since single-stranded antisense RNA did not affect cell viability (Figure 4). In the past, other studies used antisense DNA directed against the BCR/ABL fusion and were also able to demonstrate an impressive reduction of mRNA but, unfortunately, not of p210 BCR/ABL. This discrepancy was due to the fact that the antisense effect was only transient and suppression of BCR/ABL mRNA was wearing off after 8 h. Thus, BCR/ABL was considered to be a ‘difficult oncoprotein to target’ (Spiller et al., 1998) by antisense approaches. RNAi is by far more potent and enables the induction of the phenotype ‘apoptosis’ and not of a M-BCR/ABL mRNA or p210 reduction only. Given that the transfection rates were only 80%, the achieved BCR/ ABL mRNA reduction is quite impressive. A difference of 3.3 Ct values (threshold cycles) corresponds to approximately one order of magnitude. Thus, three PCR cycles later, with only 10% of non-transfected cells within the dsRNA treated population, the amount of PCR product will be similar to that in a population comprising exclusively untreated cells. However, this is Killing of leukemic cells by RNA M Wilda et al 5721 Figure 4 (a) Apoptosis in K562 cells treated with ds M-BCR/ABL, ds-M-BCR/ABL mut., antisense or sense BCR/ABL RNA as well as with STI 571. To further ensure sequence specificity of the RNAi effect, we transfected dsRNA corresponding to the mBCR/ABL fusion that did not induce apoptosis. The targeted region of m-BCR/ABL was 5’-AUGGAGACGCAGAAGCCCTT3. In addition, M-BCR/ABL dsRNA having two point mutations in its central region also lacks the apoptosis-inducing effect. The average of five independent experiments is shown, error bars indicate standard deviation, statistically significant increase of apoptosis above the controls (*P50.05), (**P50.01). (b) Morphology of K562 cells 48 h after transfection. Extensive vacuolization was seen in K562 cells treated with STI 571 and to a somewhat lesser extent in cells transfected with dsRNA targeting M-BCR/ABL but not in cells treated with either ds m-BCR/ABL or as m-BCR/ABL valid only if the 90% transfected cells show a complete BCR/ABL mRNA knockdown and are totally free of BCR/ABL mRNA. Another limitation of RNAi-targeting experiments is the transient nature of RNA transfer and the requirement for synthesis of RNA oligos before application (Tuschl, 2002). The intracellular expression of double-stranded RNA molecules from plasmid DNA is an attempt to suppress this limitation. Brummelkamp et al. (2002) used this approach to produce cells that stably suppress p53 over a period of 2 months. Incorporation of dsRNA expression Oncogene Killing of leukemic cells by RNA M Wilda et al 5722 cassettes into alternative vector systems, e.g. retroviral vectors, may also pave the way for targeting primary cells previously refractory to dsRNA treatment by liposomal transfection methods. However, one should keep in mind that the sequencespecific mRNA degradation by the RNAi is an active process that requires the proper function of a complex network of endogenous proteins. Whether the intrinsic ability to use the RNAi machinery is preserved in all cancer cells or whether cancer cells may rapidly develop a resistance to this therapeutic mRNA degradation should be addressed by future studies. One biochemical antagonistic effect to RNAi has recently been found (Scadden and Smith, 2001). The authors analysed the human adenosine deaminase that acts on dsRNA, ADAR2. In their studies, RNAi was inhibited when the dsRNA molecule was first deaminated by ADAR2. It is tempting to speculate that tumor cells may use this defense mechanism and upregulate such enzymes in response to therapeutic interventions by dsRNA. Our study further demonstrates that siRNAs are highly sequence-specific reagents and discriminate between mismatched target RNA sequences (Elbashir et al., 2001b). This predicts an additional means by which tumor cells may escape a therapeutic RNAi intervention: simply by mutation of the target fusion site. Nevertheless, during the last 2 decades the combined effort of many laboratories worldwide has led to the molecular clarification of numerous chromosomal translocations by cloning the genes involved (Rabbitts, 1994, 1998; Rowley, 1999). Silencing of these tumor-specific chimeric mRNAs by RNAi, as exemplified here using the M-BCR/ABL rearrangement, may become a promising new approach towards a molecularly targeted tumor therapy. In the near future, exploration of the RNAimediated gene therapy in mouse models (Corral et al., 1996) will provide helpful information as to whether RNAi-mediated gene therapy can really become translated into patient therapy. according to the manufacturer’s instructions. To avoid contamination with E. coli RNA, we digested the resulting plasmid DNA with DNAse-free RNAse for 1 h at 378C (Roche Diagnostics, Mannheim, Germany). The concentration of plasmid DNA was measured spectrophotometrically and copy numbers were determined according to the molecular weight of the respective inserts (for insert size see Table 1). One representative of each standard plasmid was sequenced to exclude misincorporation of single nucleotides by taq-polymerase. The plasmid standards can be obtained upon request. Quantitative PCR In the taqman PCR (taqman 7700, Perkin Elmer, Foster City, CA, USA) reactions are characterized by the point during cycling when the PCR product is first detected (the threshold cycle, Ct) rather than the amount of PCR product accumulated after a fixed number of cycles. The amounts of the various target messages, e.g. BCR/ABL, ABL, PBGD, HPRT, Lamin, and TBP were quantified by measuring Ct and by using a standard curve to determine the starting target message quantity. One principal problem for quantification of mRNA by plasmid standards is that there may be variance within the reverse transcription (RT) reaction that is not monitored during the procedure. Thus, we carefully assessed the efficiency of the cDNA synthesis by calculating the amount of cDNA after its synthesis in a set of separate experiments. Quantification of cDNA was done by Oligreen (Molecular Probes, Leiden, The Netherlands), which binds to single-stranded DNA only. When our protocol for cDNA synthesis (see below) is used, 90 – 95% of all input RNA molecules are converted into cDNA after 1 h (D Rawer, personal communication). This value was very stable and did not vary between the different target genes. For the generation of the external standard curve, we diluted the plasmid DNA in 10-fold steps, giving a range of 10 – 106 molecules. The correlation coefficients between the threshold cycle and the starting quantity of the various standard DNAs were around 0.99. Furthermore, the slope of the standard curves nearly matched the theoretical value of 73.33, (data not shown). Quantification was performed in duplicate and we observed a minimal intra-assay variation for each sample, corresponding to per cent variance of copy numbers between 3.8 and 7.9%. Materials and methods RNA isolation and cDNA synthesis Cell culture and treatment with STI 571 K562 cells were obtained from the German Collection of Microorganisms and Cell Cultures (DMSZ, Braunschweig, Germany, http://www.dsmz.de). Cells were routinely maintained in RPMI1640 medium supplemented with 10% fetal calf serum (FCS) without antibiotics in a humidified atmosphere of 5% CO2 at 378C. STI 571, Imatinib, was kindly provided by Novartis (Novartis, Switzerland). It was added at a concentration of 1 mM to exponentially growing cells. Generation of PCR standards For absolute quantification of template copy number we first cloned cDNA fragments of Lamin, M-BCR/ABL, PBGD, HPRT and the TATA-box binding protein (TBP) into the pCR II TOPO plasmid (Invitrogen, Groningen, The Netherlands). Primers used for generation of standards are shown in Table 1. Plasmids from single colonies were prepared with ion chromatography columns (PeqLab, Erlangen, Germany) Oncogene The RNA was isolated by means of a standard protocol with guanidium thiocyanate phenol-chloroform. For cDNA synthesis, we used a modified protocol which ensures that almost all RNA is converted into cDNA. Five hundred ng of total RNA was incubated with 100 ng oligo dT primers (Roche, Diagnostics), 1000 U Superscript II (Invitrogen), and 5 ml dNTP (10 nM), for 10 min at 258C followed by 50 min at 458C and 15 min at 708C. Reactions were carried out in a final volume of 100 ml containing the buffer supplied by the manufacturer (Invitrogen). As stated above, quantification of cDNA after the reverse transcription step was performed with Oligreen (Molecular Probes, Leiden, Germany) and the result was compared with the input RNA measured spectrophotometrically. Housekeeper genes for quantitative PCR We selected five housekeeper genes as endogenous RNA control and the samples were normalized on the basis of their Killing of leukemic cells by RNA M Wilda et al 5723 Table 1 Forward (FP), reverse (RP) primers and probes used for taqman PCR Target gene PCR product bp Accession Real-Time quantitative taqman PCR M BCR/ABL AJ 131466 ABL AJ 131466 TBP NM 003194 Lamin XM 002071 HPRT NM 000194 PBGD NM 000190 Probe 5’-3’: agcccttcagcggccagtagcatc FP 5’-3’: cgtccactcagccactggat RP 5’-3’: agttccaacgagcggcttc Probe 5’-3’: caacaccctggccgagttggttcat FP 5’-3’: caacactgcttctgatggcaa RP 5’-3’: cggccaccgttgaatgat Probe 5’-3’: actgttcttcactctcttggctcctgtgca FP 5’-3’: gcatattttcttgctgccagtct RP 5’-3’: accacggcactgattttcagtt Probe 5’-3’: gcttggtctcacgcagctcctcactgta FP 5’-3’: aatgatcgcttggcggtcta RP 5’-3’: aggttgctgttcctctcagcag Probe 5’-3’: ccatgttcaattatatcttccacaatcaagac FP 5’-3’: aggaaagcaaagtctgcattgtt RP 5’-3’: ggtggagatgatctctcaactttaa Probe 5’-3’: ctgttttcttccgccgttgcagc FP 5’-3’: cccacgcgaatcactctcat RP 5’-3’: tgtctggtaacggcaatgcg 104 bp Standard Calibrator clone, bp insert FP 5’-3’: tcacggatctcagcttccagatgg RP 5’-3’: ttgtgcttcatggtgatgtccgtg 1861 bp 92 bp See M-BCR-ABL 1861 bp 90 bp FP 5’-3’: cactgtttcttggcgtgtgaa RP 5’-3’: aaccaggaaataactctggctcata 1016 bp 110 bp FP 5’-3’: gcatcaccgagtctgaagaggt RP 5’-3’: tcccattgtcaatctccaccag 94 bp FP 5’-3’: aggaaagcaaagtctgcattgtt RP 5’-3’: ggtggagatgatctctcaactttaa 71 bp FP 5’-3’: aacggtggtgtgacaggcag RP 5’-3’: tgtctggtaacggcaatgcg 512 bp 94 bp 120 bp For all probes TAMRA and FAM fluorescent dyes were used as quencher or reporter, respectively. The standard plasmids were generated with the primers shown on the right side. The b-Glucoronidase gene (GUS, Accession number NM_000181) used for normalization of M-BCR/ABL copy number was amplified with the ‘ready to use’ pre-developed assay from Applied Biosystems (ABI). ABI does not provide its customers with the sequences of either primers or probe housekeeper content. The housekeeper RNA was also quantified by a standard plasmid curve. We rejected several commonly used housekeeper genes, such as b-actin, b-2 micoglobulin, and 18 S RNA, for several reasons, e.g. the existence of pseudogenes, the lack of introns or very high abundance of transcripts. Instead, we used ABL, HPRT, GUS, PDBP, and TBP, a component of the DNA-binding protein complex TFIID. To ensure RNA specificity of the taqman PCR, all primer/probe combinations were positioned over exon/intron boundaries. We then tested all primer/probe combinations using genomic DNA as template and did not observe an amplification product after 40 cycles of PCR. The taqman PCR was performed according to published protocols of the manufacturer (see http://docs.appliedbiosystems. com/). in hypotonic cell lysis buffer +350 mM NaCl, lysed by pipetting and incubated for 10 min on ice. The samples were diluted 1 : 2 with non-reducing sample buffer (1 ml: 60 ml 1 M Tris-HCl pH 6.8; 312 ml 80% Glycerol; 200 ml 10% SDS; 428 ml H2O; grains of Bromphenol blue) and electrophoresed on an 8% SDS-polyacrylamide gel. The antibodies were commercially obtained from Santa Cruz Biotechnology Inc. (Santa Cruz). In a standard Western blot protocol, for detection of the BCR/ABL fusion proteins and the ABL wild-type protein we used a rabbit polyclonal antibody against the C-terminus of c-ABL (C-19). Lamin A/ C and Vimentin antibodies were used as described previously (Elbashir et al., 2001a). The protein was detected by chemoluminescence, by means of the ECL system (Amersham, Uppsala, Sweden). Source of dsRNA molecules, transfection and luciferase assay Detection of apoptosis The dsRNA’s were commercially obtained from dharmacon (Lafayette, Co. USA). For transfection, either dsRNA or ssRNA molecules were handled exactly according to the procedure used by the Tuschl laboratory (http://www. mpibpc.gwdg.de /abteilungen / 100 / 105/ siRNAuserguide.pdf), which has also been distributed by dharmacon and summarized in their user manual (http://www.dharmacon.com/sirna.html). The dsRNA sequence for silencing the Lamin A/C mRNA and the firefly luciferase reporter gene region were used according to the work published by the Tuschl group (Elbashir et al., 2001a). The sequence of the M-BCR/ABL dsRNA as well as the oligo with two point mutations are shown in Figure 1c. Expression of firefly and sea-pansy luciferase was monitored with the Dual luciferase kit according to the manufacturer (Promega, Madison, USA) in a Berthold Luminometer (LB953, Bad Wildbad, Germany). Cells were washed with PBS (pH 7.3), resuspended and examined as cytospin preparations. We evaluated the cells morphologically in the light microscope after Wright staining. In all, five different fields were randomly selected for counting 200 cells. The percentage of apoptotic cells was calculated (Ray et al., 1994). Histone-associated DNA-fragments in the cytoplasmic fraction of cell lysates were detected by means of a sandwich-ELISA purchased from Roche (Roche Diagnostics, Mannheim, Germany). The cytoplasmic fractions of cell lysates from 56103 cells were incubated for 2 h with biotinylated antibodies directed against histones and peroxidase-coupled anti-DNA-antibodies. After removal of unbound antibodies, ABTS was added as a peroxidasesubstrate. Absorption was measured at 405 nm. Cell extraction and Western blotting Cell samples were centrifuged at 48C in a microfuge to pellet the nuclei. For nuclear extracts, the nuclei were resuspended Statistical analysis Both, values of Luciferase expression in variously transfected K562 cells and the rate of apoptosis therein were analysed by U-test according to Mann and Whitney. P values 50.05 were considered significant. Oncogene Killing of leukemic cells by RNA M Wilda et al 5724 Acknowledgments The authors wish to thank Dr T Tuschl, Dr S Viehmann and D Rawer for their help with the design of the dsRNA, quantification of BCR/ABL mRNA by taqman PCR, or generation of plasmid standards, respectively. Expert technical assistance by Stefanie Garkisch is gratefully acknowledged. A part of these studies was supported by a grant from the Deutsche Forschungsgemeinschaft to A Borkhardt. References Barthe C, Cony-Makhoul P, Melo JV and Mahon JR. (2001). Science, 293, 2163. Brummelkamp TR, Bernards R and Agami R. (2002). 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