Targeted Down-Regulation of MLL-AF9 with Antisense

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Leukemia (2001) 15, 1743–1749  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu Targeted down-regulation of MLL-AF9 with antisense oligodeoxyribonucleotide reduces the expression of the HOXA7 and -A10 genes and induces apoptosis in a human leukemia cell line, THP-1 H Kawagoe, R Kawagoe and K Sano

Department of Pediatrics, Kobe University School of Medicine, Kobe, Japan

The MLL gene is frequently rearranged in leukemias, and MLL ners are AF4 in t(4;11)(q21;q23), AF9 in t(9;11)(p22;q23), and chimeric proteins generated by chromosomal translocations ENL in t(11;19)(q23;p13).5,6 AF4, AF9, and ENL are putative play crucial roles in leukemogenesis. Targets of murine Mll include HOX proteins that regulate body pattern formation and transcription factors and these proteins activate transcription 11–13 hematopoiesis. However, it is not known whether or not the from synthetic reporter genes. Knock-in mice expressing MLL chimeric proteins regulate the HOX gene expression in Mll-AF9 under the natural Mll promoter developed leukemia, human leukemia. To address this issue, THP-1 cells, a human while the knock-in ofthe N-terminal truncated MLL fused to leukemia cell line expressing MLL-AF9, were treated with anti- a MYC-tag did not induce leukemia.14 These findings suggest sense oligodeoxyribonucleotide (ODN) complementary to the coding sequence of the MLL-AF9 junction. Down-regulation of that AF9 may play important roles in leukemogenesis as well 15 the MLL-AF9 transcript was accompanied by the reduced as N-terminus ofMLL. In another study, Dobson et al expression of the HOXA7 and -A10 genes, but not of the reported that knock-in mice expressing N-terminus ofMLL HOXA2, -A4, -A5, and -A9 genes. The number of viable cells fused to bacterial ␤-galactosidase (lacZ) developed leukemia cultured with 20 ␮M antisense ODN for 5 days was 10-fold lower with a longer latency period than MLL-AF9 knock-in mice did. than that of the sense ODN-treated control. And the number of − − They speculate that oligomerization ofthe truncated MLL by the annexin V /propidium iodide apoptotic cells in the antisense ODN-treated cells after 3 days of culture was two-fold lacZ domain may be sufficient for a dominant-negative inhi- higher than that in the control. Staining of the antisense ODN- bition ofthe wild-type MLL. Among the known MLL partner treated cells with Hoechst 33258 showed the morphology proteins, AF10 and AF17 have a leucine zipper protein dimer- characteristic to apoptosis. These results indicate that MLL- ization motifand this domain is retained in the MLL-AF10 and AF9 regulates the expression of the selected HOX genes as well MLL-AF17 fusion proteins.16,17 These results may suggest that as prevents the leukemic cells from apoptosis. Leukemia (2001) 15, 1743–1749. a dominant-negative inhibition ofwild-type MLL may be suf- Keywords: MLL-AF9; HOX; apoptosis; leukemogenesis ficient to induce leukemia and partner proteins with transcriptional regulating functions such as AF4 and AF9 facilitate this process.15 Introduction Gene-targeting studies in mice demonstrated that Mll is a positive regulator ofthe mammalian homeobox ( Hox) genes. The mixed lineage leukemia (MLL) gene (also known as ALL- A posterior shift in the anterior boundaries of the Hox genes 1, HRX, and HTRX) located at the chromosome band 11q23 expression occurs in the Mll null heterozygotes, while the is frequently rearranged by reciprocal chromosomal translo- expression ofthese genes is abolished completely in the Mll cations especially in infantile and therapy-related leukemias.1–7 null homozygotes.18 The HOX homeobox genes are originally Such translocations result in the expression ofchimeric tran- identified in Drosophila as responsible genes for homeotic scripts that are fused in frame between the MLL gene and vari- transformations.19 They encode transcription factors featured ous partner genes. The MLL gene encodes a 431-kDa protein, by a homeodomain that is a well-conserved DNA binding which comprises 3,969 amino acids.3,4 In the N-terminus motif. They are the master regulators of body patterning not region MLL protein contains three A-T hook motifs that can only in Drosophila but also in mammals.20 In mammals 39 bind DNA, and a DNA methyltransferase homology domain different HOX genes are organized into four clusters (HOXA, that possibly regulates the transcription ofits target genes. 8 -B, -C, and -D) and they are located on each different chromo- Recent study demonstrated that the A-T hook ofMLL binds some.21 Each cluster contains 9–11 genes that are classified the promoter region ofthe ARP-1 gene, that is down-regulated into 13 subgroups called ‘paralog’.22 Accumulating evidence in the Mll−/− ES cells.9 In the central and C-terminus portions indicates that the HOX genes are involved in hematopoiesis MLL has three PHD-type zinc-finger domains and a domain as well as in embryonic development.23,24 Several members for the protein interaction known as SET.3,4 These domains ofthe HOXA, -B, and -C cluster genes are expressed in human share a significant homology to Drosophila Trithorax protein hematopoietic cells and their expression patterns are closely that positively regulates the expression ofthe homeobox related to a cell lineage and stage of differentiation.25 Deregu- genes.3,4 The SET domain ofMLL interacts with hSNF5/INI1, a lation ofthe HOX genes profoundly perturbs normal hemato- component ofthe SNF/SWI complex, a chromatin remodeling poietic development.23,24 Namely, the enforced expression of system.10 The SET domain is lost when amino-terminal MLL HOXA1026 or Hoxa9 in combination with its cofactor Meisl27 fuses to partner proteins in leukemic cells. Therefore, MLL transforms murine hematopoietic cells. Thus, it is conceivable fusion proteins lack MLL functions mediated by this domain. that altering the expression ofthe HOX genes by the MLL Over 20 different translocation partners of the MLL gene aberration is important in leukemogenesis. However, it is not 5,6 have been identified to date. The most common fusion part- known whether or not the MLL chimeric proteins regulate the expression ofthe HOX genes in human leukemia. Correspondence: K Sano, 7–5–1, Chuo-ku, Kusunoki-cho, Kobe 650– In this study, we demonstrated that down-regulation of 0017, Japan; Fax: 81–78–382–6099 MLL-AF9 with antisense oligodeoxy-ribonucleotide (ODN) Received 30 April 2001; accepted 10 July 2001 reduced the expression level ofthe selected HOX genes and MLL-AF9 regulates HOX gene expression H Kawagoe et al 1744 induced apoptosis in THP-1 cells. Our results support the sequences ofthe primers forthe GAPDH transcript were: gain-of-function feature of MLL-AF9 fusion protein. forward: 5Ј-TGGTATCGTGGAAGGACTCATGAC-3Ј and reverse: 5Ј-ATGCCAGTGAGCTTCCCGTTCAGC-3Ј. The PCR condition that linearly amplified GAPDH was determined as Materials and methods described above. The optimized PCR condition was as follows: 20 cycles of94 °C for 1 min; 60°C for 1 min; 72°Cfor Leukemia cell lines 2 min. The primers used were located in separated exons to exclude the amplified products derived from contaminated THP-1,28 K56229 and HL-6030 cells were obtained from genomic DNA. The 300-bp and 180-bp PCR products of MLL- Japanese Collection ofResearch Bioresources (Tokyo, Japan). AF9 and GAPDH, respectively, were subcloned into pGEM-T These cell lines were maintained in RPMI1640 medium vector (Promega, Madison, WI, USA), sequenced, and used as containing 10% fetal calf serum (FCS). probes for Southern blot analysis. Densitometric analysis of the blot was performed by using BAS2000 image analyzing system (Fujix, Tokyo, Japan). All densitometric values were Treatment of THP-1 cells with antisense or sense divided by those of GAPDH as an internal control from the ODN against the MLL-AF9 chimeric transcript same samples, and the resulting data (normalized expression levels) were subjected to statistical analysis. Quantitativity of THP-1 is a human monocytic leukemia cell line carrying the this method is shown in Figure 1. t(9;11) chromosomal translocation. Another human leukemia cell line, HL-60, which has no t(9;11), was also used to test the specificity ofthe ODN treatment. The nucleotide Generation of representative amplified total cDNAs sequence around the breakpoint ofthe MLL and AF9 genes in THP-1 was obtained from GenBank (accession No. AJ000161), and antisense phosphorothioate ODN comp- A quantitative analysis for the HOX gene expression was performed by generating representative amplified total cDNAs lementary to the coding sequence ofthe MLL-AF9 junction Ј Ј with an oligo(dT)-based primer and poly(A)-tailing strategy as cDNA (antisense MLL-AF9, 5 -GGTTGTTCAGACTTT-3 )was 25,31 synthesized. In control experiments, the sense phosphorothio- previously described. The cDNA generated as described Ј Ј above was ethanol precipitated and resuspended in a poly(A)- ate ODN (sense MLL-AF9, 5 -AAAGTCTGAACAACC-3 ) cor- × responding to the same region was used. ODN treatments tailing solution consisting of1 terminal deoxynucleotidyl transferase (TdT) buffer (Life Technologies), 10 mm dATP were performed in triplicate by plating 1 × 104 cells in 200 ␮l (Amersham-Pharmacia Biotech), and 1.5 units/␮l TdT (Life ofRPMI1640 medium containing 2% heat-inactivated FCS ° supplemented with 20 ␮m antisense or sense ODN in wells Technologies). The sample was incubated at 37 C for 15 min and then at 70°C for 10 min. For an amplification of total ofa 48-well plate. Control cultures without adding any ODN ␮ were also prepared in triplicate. Cells were incubated at 37°C cDNA, 5 l ofthe poly(A)-tailed cDNA solution was mixed with a PCR amplification solution consisting of1 × Taq buffer, in a 5% CO incubator and the cell viability was determined 2 5mm MgCl , 0.08 ␮g/␮l 60-mer oligo-dT primer,25 1mm each by a trypan blue dye exclusion method at days 1, 3, and 5. 2 d(GCT) deoxyribonucleotide (Amersham-Pharmacia Biotech), and 0.1 units/␮l Taq DNA polymerase. PCR was performed ° RT-PCR analysis of the MLL-AF9 chimeric transcript under the following conditions: one cycle of 94 C for 1 min; 37°C for 2 min; 72°C for 10 min followed by 40 cycles of 94°C for 1 min; 55°C for 2 min; 72°C for 10 min. We also Total RNA was extracted from 1 × 104 or 2 × 104 viable cells by guanidium isothiocyanate lysis/ethanol precipitation as described.31 For reverse transcription, total RNA was resuspended in 10 ␮l ofRT solution consisting of1 × RT buffer (Life Technologies, Rockville, MD, USA), 10 mm dithiothrei- tol, 0.5 mm dNTPs (Takara, Kyoto, Japan), 25 ng/␮l oligo(dT) primer (Amersham-Pharmacia Biotech, Buckinghamshire, UK), 0.2 units/␮l RNase inhibitor (Life Technologies), and 10 units/␮l SuperScript II reverse transcriptase (Life Technologies). The sample was incubated at 42°C for 1 h, and then heated at 70°C for 10 min. For the detection of the MLL- AF9 chimeric transcripts, 1 ␮l (1/10 volume) ofthe cDNA solution was mixed with a PCR solution consisting of1 × Taq ␮ buffer (Takara), 5 mm MgCl2,2 m each primer, 1 mm dNTPs, and 0.1 unit/␮l Taq DNA polymerase (Takara). The sequences ofthe primers forthe MLL-AF9 chimeric transcript were as follows: forward primer: 5Ј-TCCAGGAAGTCAAGCAAGCA- 3Ј and reverse primer: 5Ј-TTGTTGCCTGGTCTGGGAT-3Ј. The PCR condition that linearly amplified MLL-AF9 was determined by testing the several conditions on the dilution Figure 1 Quantitative analysis of MLL-AF9 expression by RT-PCR. series ofthe cDNA generated from2 × 104 THP-1 cells. The MLL-AF9 and control GAPDH were amplified from various cellular optimized PCR condition was as follows: 23 cycles of 94°C preparations ofTHP-1 cells expressing MLL-AF9 and K562 cells, ° ° which do not express it. PCR products were analyzed by Southern for 1 min; 65 C for 1 min; 72 C for 2 min. As an internal con- blot. The expression level of MLL-AF9 normalized to GAPDH from trol, PCR detection ofthe glyceraldehyde-3-phosphate each preparation was determined by densitometric analysis, and indi- dehydrogenase (GAPDH) transcript was performed. The cated as a percentage to that oflane 1.

Leukemia MLL-AF9 regulates HOX gene expression H Kawagoe et al 1745 prepared control samples using the same procedure without adding reverse transcriptase.

Southern blot analysis of the ampliﬁed cDNA

The ampliﬁed cDNA mixtures were electrophoresed on a 1% agarose gel, denatured, and then transferred to a nylon mem- brane (Hybond-N+; Amersham-Pharmacia Biotech). Homeo- domain-free cDNA probes of HOXA2, -A4, -A7, and -A10 were generated from following ESTs (Genome Systems, St Louis, MO, USA); HOXA2: AI692874, HOXA4: T82108, HOXA7: AW629455, and HOXA10: AA213855 (numbers indicated are GenBank accession numbers). Homeodomain-

Figure 3 Quantitative analysis of HOX gene expression by representative ampliﬁcation ofcDNA. Expression of HOXA10 and ␤- actin was analyzed by Southern blot in ampliﬁed total cDNA generated from various cellular preparations of THP-1 cells expressing HOXA10, and K562 cells which do not express it. The expression level of HOXA10 normalized to ␤-actin from each preparation was determined by densitometric analysis, and indicated as a percentage to that oflane 1.

free cDNA probes of HOXA5 and -A932 were made by RT- PCR with the following primers: HOXA5: forward primer: 5Ј- AGCTGAAAAGCATGAGCATG-3Ј, and reverse primer: 5Ј- CAAGTAACACAGCTTGCTTC-3Ј, HOXA9: forward primer: 5Ј-CTGTTGATGGTAGGCTGTAT-3Ј, and reverse primer: 5Ј- AGGTGGAGAAAATGATGAAT-3Ј. Human ␤-actin cDNA probe (Clontech, Palo Alto, CA, USA) was used for an internal control. These cDNA fragments were labeled with 32P-dCTP by using a MegaPrime DNA labeling kit (Amersham-Pharma- cia Biotech) according to the manufacturer’s direction. Blots were hybridized with the radiolabeled probes at 60°C over- × night in 4.4 SSC (NaCl 0.3 m,Na3C6H5O7 0.03 m, pH 7), 7.5% formamide, 0.75% SDS, 1.5 mm EDTA, 0.75% skim milk, 370 ␮g/ml salmon sperm DNA, and 7.5% dextran sulf- ate. After hybridization, blots were washed in 0.3 × SSC, 0.1% SDS at 60°C for 30 min twice. Densitometric analysis was performed as described above with the normalization against ␤-actin as an internal control.

Apoptosis detection of ODN-treated THP-1 cells

THP-1 and HL-60 cells (3 × 104 cells) cultured with the sense or antisense ODN were washed with phosphate-buffered saline (PBS) and suspended in a binding buffer (Immunotech, Marseille, France) at 1 × 105 cells/ml. Cells were incubated with annexin V conjugated with fluorescence isothiocyanate (FITC) (Immunotech) and propidium iodide (PI) according to Figure 2 (a) MLL-AF9 expression in THP-1 cells treated with the manufacturer’s direction, and immediately analyzed on a ODN. THP-1 cells were cultured with the sense or antisense MLL- AF9 ODN for 3 days, and RT-PCR analysis of MLL-AF9 followed by flow cytometer (Epics Elite; Beckman Coulter, Marseille, Southern blot was performed. Expression of GAPDH was analyzed as France). Cells in control cultures without adding any ODN for an internal control. S, sense ODN-treated cells; AS, antisense were also analyzed by the same method. For a further assess- ODN-treatd cells. (b) Down-regulation ofthe MLL-AF9 with the anti- ment ofapoptosis, we stained the ODN-treated cells with sense ODN. Expression of MLL-AF9 and GAPDH in THP-1 cells cul- Hoechst 33258. Cells were fixed in a 1% paraformaldehyde tured with the sense or antisense ODN for 3 days was assessed by solution, washed with PBS, and incubated with 0.2 mm densitometric analysis on the blots (n = 7). The values shown indicate the mean expression levels of MLL-AF9 (normalized to GAPDH) rela- Hoechst 33258. The stained cells were observed under a flu- tive to those in the sense-treated cells. The error bar indicates a stan- orescence microscope (Zeiss, Oberkochen, Germany), and dard error (SE). *Indicates that P value was less than 0.05. the apoptotic changes ofnuclei were assessed.

Leukemia MLL-AF9 regulates HOX gene expression H Kawagoe et al 1746 Statistical analysis

The expression levels ofthe MLL-AF9 and the HOX genes normalized to those ofthe internal controls ( GAPDH and ␤-actin, respectively) were compared between the antisense-treated cells and the control sense-treated cells by Wilcoxon signed- rank test. Statistical signiﬁcance was assigned when the prob- ability that there was no difference between two variables was Ͻ0.05.

Results

Effects of the MLL-AF9 antisense ODN treatment on the HOX gene expression

To examine whether or not the MLL chimeric protein regulates the expression ofthe HOX genes, THP-1 cells expressing the MLL-AF9 transcripts were used as a model. THP-1 cells were treated with the sense or antisense ODN targeting the MLL- AF9 junction sequence. Control experiments without adding any ODN were also performed. The expression level of the MLL-AF9 transcript in the cells was analyzed by RT-PCR. The quantitative amplification of MLL-AF9 by this method is shown in Figure 1. To test it, mixtures ofvarious numbers of THP-1 cells and K562 cells, which has no t(9;11), were made. When the RT-PCR product of MLL-AF9 and GAPDH from each cellular preparation was analyzed by Southern blot, the signal intensity of MLL-AF9 normalized to that of GAPDH is linearly related to the number ofTHP-1 cells (Figure 1). After 3-day culture, 1 × 104 viable THP-1 cells were harvested and the expression of MLL-AF9 was analyzed by using this method. Representative example is shown in Figure 2a. The expression levels of MLL-AF9 normalized to those of GAPDH were compared between the sense and antisense ODN- treated cells in independent experiments (n = 7). As shown in Figure 2b, the densitometric analysis ofthe blots obtained from the independent cultures demonstrated that the expression level of MLL-AF9 in THP-1 cells treated with the antisense ODN was approximately 40% ofthat in the cells treated with the sense ODN (P Ͻ 0.05). The expression level of MLL-AF9 in THP-1 cells treated with the sense ODN was not significantly different from that in the cells cultured without ODN (data not shown). To analyze the expression of HOX genes in small numbers Figure 4 (a) Southern blot analysis ofthe amplified total cDNA ofODN-treated cells, we employed the previously described from THP-1 cells treated with the sense or antisense MLL-AF9 ODN. method for generating PCR-amplified representative total THP-1 cells were cultured with the sense or antisense MLL-AF9 ODN cDNA.25,31 The quantitative detection of HOX gene for 3 days. Amplified total cDNA was generated by RT-PCR from 1 × 104 viable cells. Each sample with and without reverse transcription expression by this method is shown in Figure 3. To test it, + − mixtures ofvarious numbers ofTHP-1 cells, which express (RT and RT , respectively) was hybridized with the specific probes for each gene and control ␤-actin. (b) Down-regulation ofthe HOXA7 HOXA10, and K562 cells, which do not express it, were and -A10 gene expression in THP-1 cells treated with the antisense made. When the amplified cDNA from each cellular prep- MLL-AF9 ODN. Expression of HOX genes and control ␤-actin in THP- aration was analyzed by Southern blot with HOXA10 and ␤- 1 cells cultured with the sense or antisense ODN for 3 days was actin probes, the signal intensity of HOXA10 normalized to assessed by densitometric analysis on the blots obtained from inde- ␤ pendent cultures (n = 7). The data represent the mean ± s.e. ofthe that of -actin is linearly related to the number ofTHP-1 cells ␤ (Figure 3). expression levels ofthe HOX genes (normalized to -actin) in the antisense ODN-treated cells relative to the control sense ODN-treated × 4 After 3-day culture, 1 10 viable THP-1 cells were har- cells. *Indicates that P value was less than 0.05. vested and the amplified cDNAs were generated. Based on the previous data ofthe HOX gene expression profile in THP- 1,33 the expression ofthe HOXA2, -A4, -A5, -A7, -A9, and (n = 7). The densitometric analysis ofthe blots obtained from -A10 genes was examined by Southern blot with the specific the independent cultures revealed that the expression levels probes for each HOX gene. A representative result is shown ofthe HOXA7 and -A10 genes were significantly decreased in Figure 4a. The expression levels ofthese HOX genes nor- in the antisense ODN-treated cells as compared to those in malized to those of ␤-actin were compared between the sense the sense ODN-treated cells (P Ͻ0.05 for both HOXA7 and and antisense ODN-treated cells in independent experiments -A10) (Figure 4b). The expression levels ofthese genes in the

Leukemia MLL-AF9 regulates HOX gene expression H Kawagoe et al 1747 sense ODN-treated cells were not significantly different from those in the cells cultured without ODN (data not shown). In contrast to HOXA7 and -A10, when the same analysis was performed on the HOXA2, -A4, -A5 and -A9 genes, the expression levels ofthese genes in the antisense ODN-treated cells were not significantly changed from those in the sense ODN-treated cells (Figure 4b). To test the specificity ofthe effect of this antisense ODN on the HOX gene expression in THP-1 cells, HL-60 cells were cultured with the same ODN (n = 6). The expression levels ofthe HOXA7 and -A10 genes in the antisense ODN-treated HL-60 cells were not significantly different from those in the cells treated with the sense ODN (data not shown). These results suggest that MLL-AF9 positively regulates the expression ofthe selected HOX genes including HOXA7 and -A10 in THP-1 cells.

Effects of the MLL-AF9 antisense ODN treatment on the growth of THP-1 cells

In a next set of experiments, the effects of the MLL-AF9 antisense ODN on the growth ofTHP-1 cells were assessed. The numbers ofthe viable THP-1 cells cultured with the antisense ODN for 3 and 5 days were four- and 10-fold lower than that ofthe sense ODN-treated cells, respectively (Figure 5a). The growth of the cells treated with the sense ODN was not different from that of the cells cultured without ODN (Figure 5a). In contrast to THP-1, such growth inhibition was not observed in HL-60 cells cultured with the same antisense ODN (Figure 5b). These results indicate that the growth inhibiting effect of this antisense ODN is specific to the cells expressing MLL- AF9. Phase-contrast microscopic observation revealed the shrinkage ofthe antisense ODN-treated cells, which is typically seen in apoptotic cells (data not shown). To detect the early apoptotic change ofthe cells, the exposition ofphosphatidylserine on the cell surface was examined with annexin V-FITC34 after 3-day culture. As shown in Figure 6a, the number ofthe annexin V +/PI− cells, which are apoptotic but not necrotic, was two-fold higher in the antisense ODN-treated cells than that in the control. The number ofthe annexin V+/PI− cells in the sense ODN-treated cells was not increased as compared to that in the cells cultured without ODN (data not shown). And when similar experiments were performed on HL-60 cells, the increase in the number ofthe apoptotic cells in the antisense ODN-treated cells was not observed Figure 5 Effects of the antisense MLL-AF9 ODN on the growth of (data not shown). For an assessment ofa later phase of THP-1 and HL-60 cells. Cells were seeded in 0.2 ml ofRPMI1640 ␮ apoptosis, the nuclear morphological changes that occurred medium containing 2% heat-inactivated FCS with 20 m ofsense ( ) or antisense (᭺) MLL-AF9 ODN. Control cultures without adding any in the ODN-treated cells were observed. Hoechst 33258 ODN were also prepared. Mean ± s.e. ofthe viable cell number ( n staining ofthe cells cultured for4 days with the antisense = 3) was plotted at days 1, 3 and 5. (a) Growth ofTHP-1 cells. ODN exhibited characteristic morphology to apoptotic cells (b) Growth ofHL-60 cells. such as chromatin condensation and nuclear segmentation (Figure 6b). Such morphological changes were not apparent in the control culture with sense ODN (Figure 6b). Together, with the antisense ODN was accompanied by the reduced these results suggest that the growth inhibitory effect of the expression of HOXA7 and -A10 in THP-1 cells while the MLL-AF9 antisense ODN is mediated, at least in part, by an expression ofother HOX genes including HOXA2, -A4, -A5, induction ofapoptosis. and -A9 was not affected (Figure 4a, b). These results suggest that MLL-AF9 positively regulates the expression ofthe HOXA7 and -A10 genes in THP-1 cells. This is the first evi- Discussion dence demonstrating that the MLL chimeric protein possibly regulates the expression ofthe HOX genes in human leukemic Accumulated evidence has indicated that the MLL chimeric cells carrying a MLL gene rearrangement. Schreiner et al35 proteins induce leukemia.14 However, their precise roles in reported that MLL-ENL strongly transactivates the promoter of leukemogenesis are not well understood. In this article, we the Hoxa7 gene in a cell-type specific manner. This transactiv- demonstrated that down-regulation ofthe MLL-AF9 transcript ation ability depends on both the methyltransferase homology

Leukemia MLL-AF9 regulates HOX gene expression H Kawagoe et al 1748 ODN against MLL-ENL induced apoptosis in KOCL33 cells. Taken together, two different MLL fusion proteins, MLL-AF9 and MLL-ENL, prevent apoptosis ofleukemic cells. Similar inhibition ofapoptosis by oncogenic chimeric proteins has been also observed in other types ofleukemias. Namely, targeted down-regulation of BCR-ABL with the antisense ODN induced apoptosis in K562 cells.40 Likewise, dominant-negative inhibition ofthe E2A-HLF protein, which is produced by the chromosomal translocation t(17;19)(q22;p13), induced apoptosis in transformed lymphocyte progenitors.41 In the case ofthe MLL-AF9 protein, such a disruption ofa cell death pathway may be a primary event in leukemogenesis. The knock-in ofthe chimeric Mll-AF9 gene causes myeloproliferation in mice as early as 6 days after birth, and then leukemia develops after a latency period of 3–7 months.14,42 This mouse study suggests that Mll-AF9 conveys a growth advantage on the myeloid progenitors. Our in vitro study supports this observation and MLL-AF9 could provide myeloid progenitors with such a growth advantage through the inhibition of apoptosis. This mechanism may allow the cells to acquire additional genetic hits for full leukemic conversion, otherwise eliminated by p53-dependent or -independent apoptotic mechanisms. In conclusion, in this article, we have demonstrated that MLL-AF9 regulates the expression ofthe selected HOX genes and prevents the cells from apoptosis in THP-1 cells. These Figure 6 (a) Increased apoptotic cell death in THP-1 cells treated effects of MLL-AF9 are likely to be important for human leuke- with the antisense MLL-AF9 ODN. THP-1 cells cultured with the sense mogenesis. However, it is possible that they are not the sole or antisense MLL-AF9 ODN for 3 days were stained with annexin V- mechanisms through which MLL-AF9 transforms cells. FITC and PI, and then analyzed on a ﬂow cytometer. Numbers indicated are the percentage ofthe cells (mean ± s.e., n = 3) in each Unknown downstream targets ofMLL-AF9 other than HOX quadrant to the total cells analyzed. Left, cells treated with sense genes may work in leukemic cells. Identiﬁcation ofsuch target ODN; right, cells treated with antisense ODN. (b) Morphological genes and understanding their biological roles will give us bet- changes ofTHP-1 cells treated with the antisense MLL-AF9 ODN. ter insights into leukemogenesis induced by MLL-AF9. The THP-1 cells cultured for 4 days with the sense or antisense MLL-AF9 antisense ODN against MLL-AF9 will be a useful tool for ODN were stained with Hoechst 33258. Left, cells treated with sense this end. ODN; right, cells treated with antisense ODN.

domain ofMLL and the C-terminus ofENL, suggesting a con- Acknowledgements tribution ofthe ENL portion to this function. 35 Since the C- terminus ofENL shows a significant homology to AF9, 35 it is This work was supported by a Grant for Priority Area and a reasonable to suppose that the AF9 portion contributes to the Grant-in-Aid for Scientific Research from the Ministry of Edu- transactivation ofthe HOX genes by the MLL-AF9 chimeric cation, Culture, Sports, Science, and Technology, Japan (to protein in a cell-type specific manner. Alternatively, it is also KS). possible to speculate that the apoptotic signal activated by the down-regulation ofMLL-AF9 caused the reduced expression ofthe HOXA7 and -A10 genes in THP-1 cells. This specu- lation does not seem to be consistent with the previous report References showing that the apoptotic signal activated by retinoic acid does not affect the expression of the 5Ј Hox genes in a mouse 1 Cimino G, Moir DT, Canaani O, Williams K, Crist WM, Katzav S, developing limb.36 However, further studies will be needed Cannizzaro L, Lange B, Nowell PC, Croce CM, Canaani E. Cloning of ALL-1, the locus involved in leukemias with the to rule out this possibility. Interestingly, both HOXA7 and t(4;11)(q21;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13) chromo- -A10 are involved in leukemogenesis. The murine Hoxa7 some translocations. Cancer Res 1991; 51: 6712–6714. gene was constitutionally activated by a retroviral insertion in 2 Ziemin-van der Poel S, McCabe NR, Gill HJ, Espinosa R, Patel Y, myeloid leukemia that outgrew in BXH-2 mouse together with Harden A, Rubinelli P, Smith SD, LeBeau MM, Rowley JD, Diaz Hoxa9 and Meis1, a binding partner of Hoxa9.37 The elevated MO. Identification ofa gene, MLL, that spans the breakpoint in expression ofthe HOXA10 gene is frequently found in human 11q23 translocations associated with human leukemias. Proc Natl 32,38 Acad Sci USA 1991; 88: 10735–10739. acute myeloid leukemia, and its enforced expression in 3 Gu Y, Nakamura T, Alder H, Prasad R, Canaani O, Cimino G, murine hematopoietic cells perturbs the myeloid differen- Croce CM, Canaani E. The t(4;11) chromosome translocation of tiation and causes leukemia in most mice transplanted with human acute leukemias fuses the ALL-1 gene, related to Droso- the transduced cells.26 Thus, the sustained expression ofthe phila trithorax, to the AF-4 gene. Cell 1992; 71: 701–708. HOXA7 and -A10 genes by MLL-AF9 may play important 4 Tkachuk DC, Kohler S, Cleary ML. Involvement ofa homologue roles in determining the transformed phenotype of THP-1 ofDrosophila Trithorax by 11q23 chromosomal translocations in acute leukemias. Cell 1992; 71: 691–700. cells. 5 Biondi A, Cimino G, Pieters R, Pui CH. Biological and therapeutic The antisense ODN against MLL-AF9 induced apoptosis in aspects ofinfantleukemia. Blood 2000; 96: 24–33. THP-1 cells (Figure 6a, b). Akao et al39 reported that antisense 6 DiMartino JF, Cleary ML. MLL Rearrangements in haematological

Leukemia MLL-AF9 regulates HOX gene expression H Kawagoe et al 1749 malignancies: lessons from clinical and biological studies. Br J Reid DS, Largman C, Lawrence HJ, Humphries RK. Differential Haematol 1999; 106: 614–626. expression ofhomeobox genes in functionallydistinct CD34 + sub- 7 Pui CH, Relling MV, Rivera GK, Hancock ML, Raimondi SC, Hes- populations ofhuman bone marrow cells. Proc Natl Acad Sci USA lop HE, Santana VM, Ribeiro RC, Sandlund JT, Mahmoud HH, 1994; 91: 12223–12227. Evans WE, Crist WM, Krance RA. Epipodophyllotoxin-related 26 Thorsteinsdottir U, Sauvageau G, Hough MR, Dragowska WH, acute myeloid leukemia: a study of35 cases. Leukemia 1995; 9: Lansdorp PM, Lawrence HJ, Largman C, Humphries RK. Over- 1990–1996. expression of HOXA10 in murine hematopoietic cells perturbs 8 Zeleznik-Le NJ, Harden AM, Rowley JD. 11q23 translocations split both myeloid and lymphoid differentiation and leads to acute the ‘AT-hook’ cruciform DNA-binding region and the transcrip- myeloid leukemia. Mol Cell Biol 1997; 17: 495–505. tional repression domain from the activation domain of the mixed- 27 Kroon E, Krosl J, Thorsteinsdottir U, Baban S, Buchberg AM, Sau- lineage leukemia (MLL) gene. Proc Natl Acad Sci USA 1994; 91: vageau G. Hoxa9 transforms primary bone marrow cells through 10610–10614. specific collaboration with Meis 1a but not Pbx 1b. EMBO J 1998; 9 Arakawa H, Nakamura T, Zhadanov AB, Fidanza V, Yano T, 17: 3714–3725. Bullrich F, Shimizu M, Blechman J, Mazo A, Canaani E, Croce 28 Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, CM. Identification and characterization ofthe ARP1 gene, a target Tada K. Establishment and characterization ofa human acute for the human acute leukemia ALL1 gene. Proc Natl Acad Sci USA monocytic leukemia cell line (THP-1). Int J Cancer 1980; 26: 1998; 95: 4573–4578. 171–176. 10 Rozenblatt-Rosen O, Rozovskaia T, Bukarov D, Sedkov Y, Tillib 29 Lozzio CB, Lozzio BB. Human chronic myelogenous leukemia S, Blechman J, Nakamura T, Croce CM, Mazo A, Canaani E. The cell-line with positive Philadelphia chromosome. Blood 1975; 45: C-terminal SET domains ofALL1 and TRITHORAX interact with 321–334. the INI1 and SNR1 proteins, components ofthe SWI/SNF com- 30 Gallagher RE, Collins SJ, Trujillo J, McCredie K, Ahearn M, Tsai plex. Proc Natl Acad Sci USA 1998; 95: 4152–4157. S, Metzgar R, Aulakh G, Ting R, Ruscetti F, Gallo RC. Characteriz- 11 Morrissey JJ, Raney S, Cleary ML. The FEL (AF-4) protein donates ation of the continuous differentiating myeloid cell line (HL-60) transcriptional activation sequences to HRX-FEL fusion proteins in from a patient with acute promyelocytic leukemia. Blood 1979; leukemias containing t(4;11)(q21;q23) chromosomal translo- 54: 713–733. cations. Leukemia Res 1997; 21: 911–917. 31 Brady G, Barbara M, Iscove NN. Representative in vitro cDNA 12 Prasad R, Yano T, Sorio C, Nakamura T, Rallapalli R, Gu Y, Lesh- amplification from individual hematopoietic cells and colonies. kowitz D, Croce CM, Canaani E. Domains with transcriptional Meth Mol Cell Biol 1990; 2: 17–25. regulatory activity within the ALL1 and AF4 proteins involved in 32 Kawagoe H, Humphries RK, Blair A, Sutherland HJ, Hogge DE. acute leukemia. Proc Natl Acad Sci USA 1995; 92: 12160–12164. Expression ofHOX genes, HOX-cofactors,and MLL in pheno- 13 Rubnitz JE, Morrissey J, Savage PA, Cleary ML. ENL, the gene typically and functionally defined subpopulations of leukemic and fused with HRX in t(11;19) leukemias, encodes a nuclear protein normal human hematopoietic cells. Leukemia 1999; 13: 687–698. with transcriptional activation potential in lymphoid and myeloid 33 Vieille-Grosjean I, Roullot V, Courtois G. Lineage and stage spe- cells. Blood 1994; 84: 1747–1752. cific expression ofHOX 1 genes in the human hematopoietic sys- 14 Corral J, Lavenir I, Impey L, Warren AJ, Forster A, Larson TA, Bell tem. Biochem Biophys Res Commun 1992; 183: 1124–1130. S, McKenzie ANJ, King G, Rabbitts TH. An Mll-Af9 fusion gene 34 Vermes I, Haanen C, Reutelingsperger CPM. A novel assay for made by homologous recombination causes acute leukemia in apoptosis: flow cytometric detection ofphosphatidylserine chimeric mice: a method to create fusion oncogenes. Cell 1996; expression on early apoptotic cells using fluorescein labeled 85: 853–861. annexin V. J Immunol Meth 1995; 180: 39–52. 15 Dobson CL, Warren AJ, Pannell R, Forster A, Rabbitts TH. Tumo- 35 Schreiner SA, Garciá-Cue´llar, MP, Fey GH, Slany RK. The leukem- rigenesis in mice with a fusion of the leukaemia oncogene Mll ogenic fusion of MLL with ENL creates a novel transcriptional and the bacterial lacZ gene. EMBO J 2000; 19: 843–851. transactivator. Leukemia 1999; 13: 1525–1533. 16 Chaplin T, Ayton P, Bernard OA, Saha V, Della Valle V, Hillion 36 Dupe´ V, Ghyselinck NB, Thomazy V, Nagy L, Davies PJA, Cham- J, Gregorini A, Lillington D, Berger R, Young BD. A novel class bon P, Mark M. Essential roles ofretinoic acid signaling in inter- ofzinc finger/leucine zipper genes identified fromthe molecular digital apoptosis and control ofBMP-7 expression in mouse auto- cloning ofthe t(10;11) translocation in acute leukemia. Blood pods. Dev Biol 1999; 208: 30–43. 1995; 85: 1435–1441. 37 Nakamura T, Largaespada DA, Shaughnessy JD Jr, Jenkins NA, 17 Prasad R, Lesshkowitz D, Gu Y, Alder H, Nakamura T, Saito H, Copeland NG. Cooperative activation ofHoxa and Pbx1-related Huebner K, Berger R, Croce CM, Canaani E. Leucine-zipper genes in murine myeloid leukemias. Nat Genet 1996; 12: 149– dimerization motifencoded by the AF17 gene fusedto ALL-1 153. (MLL) in acute leukemia. Proc Natl Acad Sci USA 1994; 91: 38 Lawrence HJ, Sauvageau G, Ahmadi N, Lopez AR, LeBeau MM, 8107–8111. Link M, Humphries K, Largman C. Stage- and lineage-specific 18 Yu BD, Hess JL, Horning SE, Brown GAJ, Korsmeyer SJ. Altered expression ofthe HOXA10 homeobox gene in normal and leu- Hox expression and segmental identity in Mll-mutant mice. kemic hematopoietic cells. Exp Hematol 1995; 23: 1160–1166. Nature 1995; 378: 505–508. 39 Akao Y, Mizoguchi H, Misiura K, Stec WJ, Seto M, Ohishi N, Yagi 19 Gehring WJ. Homeoboxes in the study ofdevelopment. Science K. Antisense oligodeoxyribonucleotide against the MLL-LTG19 1987; 236: 1245–1252. chimeric transcript inhibits cell growth and induces apoptosis in 20 Kessel M, Gruss P. Murine developmental control genes. Science cells ofan infantileleukemia cell line carrying the t(11;19) 1990; 249: 374–379. chromosomal translocation. Cancer Res 1998; 58: 3773–3776. 21 Graham A, Papalopulu N, KrumlaufR. The murine and Droso- 40 McGahon A, Bissonnentte R, Schmitt M, Cotter KM, Green DR, phila homeobox genes complexes have common features of Cotter TG. BCR-ABL maintains resistance ofchronic myelogene- organization and expression. Cell 1989; 57: 367–378. ous leukemia cells to apoptotic cell death. Blood 1994; 83: 22 Scott MP. Vertebrate homeobox gene nomenclature. Cell 1992; 1179–1187. 71: 551–553. 41 Inaba T, Inukai T, Yoshihara T, Seyschab H, Ashmun RA, Canman 23 Lawrence HJ, Sauvageau G, Humphries RK, Largman C. The role CE, Laken SJ, Kastan MB, Look T. Reversal ofapoptosis by the ofhomeobox genes in normal and leukemic hematopoiesis. Stem leukaemia-associated E2A-HLF chimaeric transcription factor. Cells 1996; 14: 281–291. Nature 1996; 382: 541–544. 24 van Oostveen JW, Bijl JJ, Raaphorst FM, Walboomers JJM, Meijer 42 Dobson CL, Warren AJ, Pannell R, Forster A, Lavenir I, Corral J, CJLM. The role ofHomeobox genes in normal hematopoiesis and Smith AJH, Rabbitts TH. The Mll-AF9 gene fusion in mice controls hematological malignancies. Leukemia 1999; 13: 1675–1690. myeloproliferation and specifies acute myeloid leukaemogenesis. 25 Sauvageau G, Lansdorp PM, Eaves CJ, Hogge DE, Dragowska WH, EMBO J 1999; 18: 3564–3574.

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