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Dr M. Satake, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan. E-mail: email@example.com
Human acute myeloid leukaemia (AML) involving a core-binding factor (CBF) transcription factor is called CBF leukaemia. In these leukaemias, AML1 (RUNX1, PEBP2αB, CBFα2)-MTG8 (ETO) and CBFβ (PEBP2β)-MYH11 chimaeric proteins are generated by t(8;21) and inv(16) respectively. We analysed gene expression profiles of leukaemic cells by microarray, and selected genes whose expression appeared to be modulated in association with t(8;21) and inv(16). In a pair-wise comparison, 15% of t(8;21)-associated transcripts exhibited high or low expression in inv(16)-AML, and 26% of inv(16)-associated transcripts did so equivalently in t(8;21)-AML. These common elements in gene expression profiles between t(8;21)- and inv(16)-AML probably reflect the situation that AML1-MTG8 and CBFβ-MYH11 chimaeric proteins affect a common set of target genes in CBF leukaemic cells. On the other hand, 38% of t(8;21)-associated and 24% of inv(16)-associated transcripts were regulated in t(8;21)- and inv(16)-specific manners. These distinct features of t(8;21)- and inv(16)-associated genes correlate with the bimodular structures of the chimaeric proteins (CBF-related AML1 and CBFβ portions, and CBF-unrelated MTG8 and MYH11 portions).
Human acute myeloid leukaemia (AML) is a heterogeneous group of diseases. Diagnosis of AML-subtypes is of clinical importance, as they vary in their responsiveness to therapy and prognosis. These subtypes are recognised by the morphology of leukaemic cells and classified using the French–American–British (FAB) system. Each FAB subtype corresponds to the differentiation blockage of leukaemic cells at a specific stage in a certain lineage. In addition, various types of chromosomal rearrangements are seen in AML, and a particular type of chromosomal translocation is sometimes associated with a FAB subtype. Among AML-subtypes, genes most frequently encountered in the clinic are those encoding a core-binding factor (CBF; PEBP2) transcription factor (Look, 1997; Speck & Gilliland, 2002). AML involving CBF is known as CBF leukaemia. The AML1 (RUNX1, PEBP2αB, CBFα2) and CBFβ (PEBP2β) proteins constitute both DNA-binding and non-binding subunits of a heterodimeric CBF. Due to chromosomal rearrangements, the AML1-MTG8 (ETO) and CBFβ-MYH11 chimaeric genes are generated in t(8;21)-AML with an M2 subtype and in inv(16)-AML with an M4Eo subtype respectively (Miyoshi et al, 1991, 1993; Erickson et al, 1992; Liu et al, 1993).
Functional studies using reporter assays indicate that the AML1-MTG8 and CBFβ-MYH11 chimaeric polypeptides exhibit dominant negative activity against CBF-dependent transcription (Peterson & Zhang, 2004; Shigesada et al, 2004). Furthermore, when cDNAs of AML1-MTG8 and CBFβ-MYH11 are knocked into the respective Aml1 and Cbfβ loci, knocked-in heterozygotes exhibit essentially similar phenotypes as those seen in Aml1 (−/−) and Cbfβ(−/−) mice (Castilla et al, 1996; Yergeau et al, 1997). These phenotypes do not include leukaemia but rather involve the failure to develop definitive-type haematopoiesis, in strong support of the hypothesis that chimaeric proteins function as dominant negatives. This observation suggests that the two chimaeric proteins modulate expression of a common set of target genes in CBF leukaemic cells.
Alternatively, AML1-MTG8 and CBFβ-MYH11 proteins could also modulate gene expression in a specific fashion, as the amino acid sequence of C-terminal portion of each chimaeric protein is not homologous to CBF or to the other. MTG8 belongs to the Nervy family proteins (Kitabayashi et al, 1998) and reportedly interacts with various factors including transcriptional co-repressors (Peterson & Zhang, 2004). On the other hand, the MYH11 portion of the chimaera represents a rod domain seen in smooth muscle myosin heavy chain protein.
Several attempts to correlate karyotypic classification of AML with the gene expression profiles have been reported. Such studies have focused on t(8;21), t(15;17), inv(16), 11q23-alteration and normal karyotypes (Schoch et al, 2002; Debernardi et al, 2003; Kohlmann et al, 2003; Bullinger et al, 2004; Ross et al, 2004; Gutierrez et al, 2005). A recent report classified all manifestations of AML into 16 distinct subgroups based on gene expression profiles (Valk et al, 2004). Given that the AML1-MTG8 and CBFβ-MYH11 proteins are structurally bimodular, this study aimed to examine whether these chimaeric proteins regulate common as well as unique sets of targets in CBF leukaemia. To do so, we analysed gene expression profiles of AML clinical samples by microarray and found that several genes were commonly regulated in t(8;21)- and inv(16)-AML, and that others were regulated in t(8;21)-specific and inv(16)-specific manners. Below we describe our approach and discuss implications of our results.
Materials and methods
A total of 50 paediatric AML patients were enroled in this study (Table SI). Among these, 45 were derived from 54 patients reported previously (nine of the 54 patients were excluded because their FAB subtype or karyotype information could not be obtained) (Yagi et al, 2003). The other five patients (U06, U09, U11, U20 and U21) were included in this study to increase the number of samples grouped as G2, G3 and G4 (see below). Four normal bone marrow samples enroled in this study were also described previously (Yagi et al, 2003). This study was approved by the ethics committee of the National Cancer Centre and conducted according to tenets of the Declaration of Helsinki. Informed consent was obtained from each patient.
Microarray and statistical analysis
The microarray used in this study was Human Genome U95Av2 (Affymetrix, Santa Clara, CA, USA) containing 12 566 probe sets. For the 45 previously reported samples, the scanned image data obtained previously (Yagi et al, 2003) were re-used. Microarray analysis of the newly included samples was performed as described (Yagi et al, 2003). The analysis included preparation of mononuclear cells from bone marrow or peripheral blood, total RNA isolation, monitoring RNA integrity, preparation of biotin-labelled cRNA from total RNA, hybridisation to the microarray, and washing, staining and scanning of samples. Scanned image data were processed using Affymetrix Microarray Suite software version 5.0, and an expression value (signal) of each probe set was calculated and normalised, such that the mean of signal values in each experiment was 100, to adjust for minor differences between experiments. Statistical analyses and fold change calculations were performed using expression values that were log-transformed after the addition of 10. Hierarchical clustering analysis and matrix presentation were performed using cluster and tree view software.
To validate selected genes, microarray data of adult AML reported by Valk et al (2004) were down-loaded from the NCBI Gene Expression Omnibus database (GSE 1159 in http://www.ncbi.nlm.nih.gov/projects/geo/), and the data of 222 AML and eight normal samples were used after excluding data from 57 samples whose FAB subtype or karyotype information was not given. Promoter analysis, including prediction of a transcription initiation site, extraction of genomic sequence around the initiation site, and assignment of transcription factor-binding sites, was performed using the genomatixsuite software (Genomatix, Munich, Germany). To assign AML1-, POU4F1-, and HOXB2-binding sites, V$AML1.01, V$BRN3.01, and V$HOXA9.01 matrixes in the software were used respectively.
For semi-quantitative RT-PCR analysis, cDNA was prepared from 0·5 μg of total RNA, and one fiftieth of the cDNA was used as template for each PCR reaction. Forward and reverse primers were designed using genetyx mac 9.0/search primer software. Sequences of forward and reverse primers were: CD34, 5′-ATTTCCTGATGAATCGCCGC-3′ and 5′-GCCTTTCCCTGAGCCTCAGG-3′; CAV1, 5′-ACCTCAACGATGACGTGGTC-3′ and 5′-CAAGTTGATGCGGACATTGC-3′; CLIPR-59, 5′-GTCTTCGCACCAGCATCCCG-3′ and 5′-AGGTTTCTGATCCAGGGTTG-3′; and HOXA9, 5′-GCACCGCTTTTTCCGAGTG-3′ and 5′-GCGGTGTACCACCACCATC-3′. PCR using Amplitaq Gold (Applied Biosystems, Foster City, CA, USA) was performed using conditions appropriate for each transcript. PCR products were run on agarose gels.
Selection of t(8;21)- and inv(16)-associated genes
A goal of this study was to extract genes whose expression was modulated in the presence of t(8;21) and inv(16) and evaluate how many genes were similarly up or downregulated in t(8;21)- and inv(16)-AML. For this purpose, we used microarray gene expression data of 50 paediatric AML patients including eight cases of t(8;21) and seven cases of inv(16). Initially, unsupervised hierarchical clustering analysis was performed as shown in Fig 1, in which samples are identified together with their FAB-subtype. All eight t(8;21) samples and six of seven inv(16) samples (except sample S12) were clustered as distinct groups, indicating that t(8;21)- and inv(16)-AML constitute an independent AML subgroup. In addition, neighbouring of t(8;21) and inv(16) clusters suggests that there may exist a common element in their gene expression.
To extract t(8;21)- and inv(16)-associated genes, we first categorised AML samples into five groups, G1–G5, according to FAB subtype and karyotype [G1, M2 with t(8;21); G2, other M2; G3, M4 with inv(16); G4, other M4; and G5, other FAB subtypes]. Sample numbers in G1, G2, G3, G4 and G5 were 8, 5, 7, 7 and 23 respectively. The method of gene extraction employed is shown schematically in Fig 2A. First, G1 was compared with G2 and also with a combined group of G2 + G4 + G5 and extracted transcripts whose average expression in G1 was more than twofold higher with a P < 0·01 according to the Student's t-test. As both G1 and G2 belong to the same M2-subtype, transcripts extracted by comparing G1 and G2 are considered to represent those associated with the t(8;21) abnormality but not with the M2-subtype. Transcripts extracted by these two rounds of comparison were defined as t(8;21)-associated highly expressed transcripts. Similarly, inv(16)-associated highly expressed transcripts were extracted by two rounds of comparison between G3 and G4, and between G3 and the G2 + G4 + G5 group. t(8;21)- and inv(16)-associated low expression transcripts, whose average expression was more than twofold lower with a P < 0·01, were also extracted. G3 samples were not included to extract t(8;21)-associated transcripts, and G1 samples were not used to extract inv(16)-associated transcripts. Thus, t(8;21)- and inv(16)-associated genes were selected independently of each other. As summarised in Fig 2B, 59 t(8;21)-associated highly expressed transcripts, 58 inv(16)-associated highly expressed transcripts, 15 t(8;21)-associated low expression transcripts, and 18 inv(16)-associated low expression transcripts were selected.
Selection of commonly and specifically regulated genes
The gene extraction method shown in Fig 2A identified six highly expressed and two low expression transcripts associated with both t(8;21) and inv(16) (Fig 2B). A review of the extracted genes, however, suggested that the numbers of commonly regulated transcripts was probably underestimated. For example, 19 of the 58 inv(16)-associated highly expressed transcripts showed more than twofold greater expression not only in G3 but also in G1 (data not shown). This is not surprising, as the extraction procedure described above adopted the strict standard of P < 0·01. Therefore, we modified the gene extraction procedure as shown in Fig 2C with the goal of selecting transcripts both commonly and specifically up or downregulated in the presence of t(8;21)/inv(16). Therefore, in the common group, transcripts showing more than a twofold increase or decrease in expression at P < 0·05 rather than P < 0·01 were added to the previously described six and two common transcripts. Thus, 17 and 6 transcripts were defined as commonly increased and decreased in expression respectively. In addition, transcripts whose average expression in G3 was more than 1·25-fold higher compared with G4 or G2 + G4 + G5 were excluded from the t(8;21)-associated highly expressed transcripts, and the remaining 25 transcripts were defined as t(8;21)-specific highly expressed transcripts. Using similar modifications, inv(16)-specific highly expressed transcripts (n = 15), t(8;21)-specific low expression transcripts (n = 3), and inv(16)-specific low expression transcripts (n = 3) were defined.
According to our criteria, 8 of 59 t(8;21)-associated highly expressed transcripts were also highly expressed in inv(16)-AML, and 3 of the 15 t(8;21)-associated low expression transcripts were also low expression transcripts in inv(16)-AML. Similarly, 15 of 58 inv(16)-associated highly expressed transcripts were also highly expressed in t(8;21)-AML, and five of 18 inv(16)-associated low expression transcripts were also of low expression in t(8;21)-AML. Overall, 15% (11/74) of t(8;21)-associated transcripts exhibited similar expression in inv(16)-AML, and 26% (20/76) of inv(16)-associated transcripts did so in t(8;21)-AML. On the other hand, 38% (25 + 3/74) of t(8;21)-associated and 24% (15 + 3/76) of inv(16)-associated transcripts appeared to be regulated in t(8;21)- and inv(16)-specific manners respectively. In summary, these results indicate that there exists a significant number of commonly regulated transcripts in addition to those regulated specifically by either t(8;21)- or inv(16)-AML. Although the genes whose expression was unique to either t(8;21)- or inv(16)-AML have been reported repeatedly, common gene expression signatures to both AML-subtypes are the first demonstration.
It must be noted that in some cases multiple probe sets were used for a specific gene. Taking redundancy into account, the numbers of individually selected genes were as follows: 15 common, 21 t(8;21)-specific and 13 inv(16)-specific genes as highly expressed; and 6 common, 3 t(8;21)-specific and 3 inv(16)-specific genes as low expressed. Table I details a list of these genes and includes the ratios of average expression values.
Table I. Transcripts whose expression was modulated commonly to t(8;21)- and inv(16)-AML and specifically to each.
G1/G2 + 4 + 5
G3/G2 + 4 + 5
Values of ratio highlighted by pink and blue represent more than twofold high and low expression respectively.
Mitogen-activated protein kinase-activated protein kinase 3
Phospholipid transfer protein
Proline-rich nuclear receptor coactivator 1
POU domain, class 4, transcription factor 1
POU domain, class 4, transcription factor 1
RAS p21 protein activator 4
Runt-related transcription factor 1/MTG8
Serine (or cysteine) proteinase inhibitor, member 2
SHC transforming protein 1
Solute carrier family 25, member 1
Solute carrier family 25, member 1
Solute carrier family 25, member 1
Vascular endothelial growth factor
Vascular endothelial growth factor
Chromosome 6 open reading frame 145
C-type lectin domain family 10, member A
Cysteine-rich motor neuron 1
Chemokine (C-X-C motif) ligand 2
Homeo box B2
Integrin, beta 5
Myosin, heavy polypeptide 11, smooth muscle
Myosin, heavy polypeptide 11, smooth muscle
5’-nucleotidase, ecto (CD73)
src family associated phosphoprotein 1
Secreted phosphoprotein 1 (osteopontin)
Secreted phosphoprotein 1 (osteopontin)
Transmembrane 4 superfamily member 1
Transmembrane 4 superfamily member 1
Adenylate cyclase 7
Homeo box A10
Homeo box A5
Homeo box A9
CD33 antigen (gp67)
Excision repair deficiency, complementation group 8
Protein tyrosine phosphatase, non-receptor type 12
Core-binding factor, beta subunit
Cytoplasmic linker 2
Evaluation of selected genes
Expression data of selected genes were processed for matrix presentation, as shown in Fig 3. For this analysis samples and probe sets were aligned by alphabetical and numerical order respectively, within the each group. Each member of G1 and G3 groups was clearly separated from other groups in terms of the expression of selected genes. Also, t(8;21)-specific and inv(16)-specific as well as commonly regulated genes, showed patterns unique to the relevant groups, indicating that the method of gene extraction functioned effectively.
We next investigated whether the expression evaluated by the microarray analysis reflected RNA levels. To do so, four representative transcripts were chosen: CD34 from the common highly expressed group, CAV1 from t(8;21)-specific highly expressed genes, CLIPR-59 from inv(16)-specific highly expressed genes, and HOXA9 from the common low expression group. cDNA was synthesised from RNA from 17 samples and processed for semi-quantitative RT-PCR (Fig 4). Overall the relative level of each transcript in a respective sample paralleled the relative expression values obtained by microarray analysis (see also the legend to Fig 4).
A control analysis was performed to evaluate the significance of common gene expression signatures that were found in t(8;21)- and inv (16)-AML. This was done by examining how many genes might be selected as commonly regulated between G2 and G4 subgroups. Each of G2 and G4 were used as test samples, and G1, G3 and G1 + G3 + G5 were used as reference samples. The gene extraction procedure used was (Fig S1) similar to that shown in Fig 2, and the selected genes are listed in Table SII. Only three transcripts (two genes) were selected as commonly high expressed, and no transcript was extracted as commonly low expressed (5 G2-specific and 26 G4-specific highly expressed transcripts and 10 G2-specific and 11 G4-specific low expression transcripts were selected at the same time). The gene number common to G2 and G4 was much smaller compared with that common to t(8;21) and inv(16) [23 transcripts (21 genes)]. This indicates the following: firstly, very few common elements exist in gene expression of G2- and G4-subgroups. Secondly, the above described 23 transcripts were not selected by chance but probably reflect common features of t(8;21)- and inv(16)-AML.
Validation of selected genes using a different set of microarray data
We next evaluated whether the selected genes were valid indicators of t(8;21)- and inv(16)-AML activities. To do so we employed another set of microarray data from AML patients reported by Valk et al (2004). Their data set consisted of 285 patients and contained information on FAB-classification and karyotype for each AML sample. We used the data of 222 of those patients and excluded the other 63 due to lack of FAB subtype and karyotype information. According to our classification, the numbers of AML samples were 20 in G1, 35 in G2, 14 in G3, 31 in G4 and 122 in G5.
Figure 5 shows a matrix presentation of the expression data. Each of the aforementioned six categories of genes again appeared to behave as a distinct cluster, suggesting that most genes selected were indeed modulated in the presence of t(8;21) and inv(16) activities. Genes selected as inv(16)-specific highly expressed, however, did not yield such reproducible results. Half appeared to be high in the G3 samples, whereas the other half did not. Although the reason for this discrepancy is not clear, the samples used in our study were from paediatric patients, whereas those in Valk et al (2004) were from adults. In the case of inv(16)-AML, factors regulating gene expression may differ between childhood and adulthood AML (see Fig 5 legend regarding the comparison of our analysis and that of Valk et al (2004).
Analysis of AML1-binding sites in a promoter region
Finally, we investigated whether the promoter region of a selected gene harbors AML1-binding sites. Only highly expressed genes were examined, as the number of low expression genes was relatively small. Putative AML1-binding sites were evaluated in a genomic region covering −2000 to +500 bp with respect to the predicted transcription initiation site, using genomatixsuite software. We determined the number of AML1-binding sites for a specific promoter and calculated the average ± standard deviation for highly expressed genes of the common, t(8;21)-specific and inv(16)-specific groups (Table II). As references, 44 and 46 probe sets were used whose expression values showed approximately the average and median respectively, of all probe sets on the microarray. As seen in Table II, a significant difference was not detected in the average number of AML1-binding sites between promoters of selected genes and those of the reference genes. POU4F1 and HOXB2, which encode transcription factors, were extracted as a t(8;21)-specific highly expressed gene and an inv(16)-specific highly expressed gene respectively. The numbers of POU4F1- and HOXB2-binding sites in t(8;21)-specific and inv(16)-specific highly expressed genes were the same as those seen in the reference genes. This result suggests that the number of putative AML1-, POU4F1- and HOXB2-binding sites in the promoter region cannot account for the expression levels of the extracted genes.
Table II. Number of transcription factor-binding sites per gene.
No. of genes
Transcription factor-binding sites were predicted using the genomatixsuite software l. The average number of sites per gene was calculated and presented together with SD for each group of genes.
Commonly high expressed (A)
1·60 ± 1·66
3·07 ± 2·57
1·13 ± 0·88
t(8;21)-specifically high expressed (B)
1·24 ± 1·11
2·33 ± 2·47
1·56 ± 1·83
inv(16)-specifically high expressed (C)
1·10 ± 1·30
2·10 ± 2·02
2·10 ± 1·70
A + B + C
1·33 ± 1·39
2·57 ± 2·46
1·55 ± 1·59
1·64 ± 3·03
3·50 ± 4·60
2·68 ± 4·40
1·43 ± 1·33
4·37 ± 5·25
1·59 ± 2·00
To compare gene expression between t(8;21)- and inv(16)-AML, AML samples were classified as groups G1–G5 and a pair-wise comparison was performed between these groups. Because of the combinations of test and reference samples used here, the genes identified are probably associated with t(8;21) and inv(16), but not with the lineage/stage specificity of leukaemic cells as exemplified by the FAB-classification. The existence of a gene expression signature characteristic of CBF leukaemia was reported previously (see Fig 4 in Ross et al, 2004, although a common element in t(8;21) and inv(16) is not immediately clear). However, our approach is unique in that the t(8;21)- and inv(16)-associated genes were selected independently of inv(16)- and t(8;21)-AML respectively, and commonly modulated genes were selected by comparing t(8;21)- and inv(16)-associated genes. Thus, we demonstrate that t(8;21)- and inv(16)-AML exhibit significant overlap in gene expression signatures. This result indicates that AML1-MTG8 and CBFβ-MYH11 chimaeric proteins affect a common set of targets in leukaemic cells. In addition, our method identifies new genes not previously reported.
Detection of commonly regulated genes agrees with the notion that both AML1-MTG8 and CBFβ-MYH11 exert a similar dominant negative effect on wild type CBF. Furthermore, our identification of specifically regulated genes is in line with the prediction that each chimaeric protein may also have a unique activity. Promoter analysis of selected genes, however, suggests that regulation is complex. The number of putative AML1-binding sites in a predicted promoter region did not differ significantly between selected and reference genes, regardless of the type of genes selected as common or specific highly expressed genes. This observation was also true for the POU4F1- and HOXB2-binding sites for t(8;21)- and inv(16)-specific highly expressed gene promoters respectively. Several mechanisms could explain the relationship between transcriptional activity of a chimaeric protein and regulation of gene expression. One is that these proteins regulate promoters through sites other than AML1 binding sites by interacting with other transcription factors and/or co-factors. For example, AML1-MTG8 interacts with p300/CBP and interferes with transcription mediated by an E-box transcription factor whose binding site is distinct from that of AML1 (Zhang et al, 2004). Alternatively, although both chimaeric proteins could target the promoters of the same set of genes, environmental factors, such as cell lineage or developmental stage, that influence the magnitude of gene expression may vary for each chimaera. A chimaeric protein may also target an unidentified molecule, which in turn could modulate expression of extracted genes.
Of selected genes, MTG8 and MYH11 are of interest. The probe sets for these genes correspond to the MTG8 portion of the AML1-MTG8 chimaeric transcript and to the MYH11 portion of the CBFβ-MYH11 chimaeric transcript respectively. The fact that the probe sets for MTG8 and MYH11 were selected as t(8;21)- and inv(16)-specific highly expressed genes respectively, indicates that our gene extraction procedure worked efficiently. Expression of MTG8 and MYH11 have been assigned repeatedly as the most characteristic signatures of t(8;21) and inv(16) respectively, in other microarray analyses (Schoch et al, 2002; Debernardi et al, 2003; Kohlmann et al, 2003; Bullinger et al, 2004; Ross et al, 2004; Valk et al, 2004; Gutierrez et al, 2005). Immunophenotyping studies of t(8;21)- and inv(16)-leukaemic cells document CD34 and HLA-DR as specific markers that may reflect the immaturity of cells (Hurwitz et al, 1992; Osato et al, 1997). Identification of corresponding transcripts as common highly expressed genes is probably the basis of this immunophenotype of cells. High expression of CCND2 also may confer a growth advantage on leukaemic cells, although ectopic expression of CBFβ-MYH11 in 32Dcl3 and Ba/F3 cell lines has been reported to retard the G1 to S transition and stimulate expression of p21WAF1 (Cao et al, 1997; Lou et al, 2000). It also is noteworthy that many genes related to endothelial cells and signal transduction were extracted as t(8;21)-specific highly expressed genes. The endothelial group includes VEGF, FBLN5, and ITGB4, whereas the genes that encoded signalling factors were VEGF, ADRA2C, SHC1, CAV1, RASA4, ARHGEF6 and MAPKAPK3. Also potentially critical are genes that were extremely highly expressed, including TRH, TPSAB1, MN1, POU4F1 and CAV1.
Among the low expression genes, HOXA5, HOXA9 and HOXA10 were categorised as common genes. Enhanced expression of HOXA9 is well established in many AML-subtypes including those with normal karyotypes (Lawrence et al, 1999; Debernardi et al, 2003; Bullinger et al, 2004; Gutierrez et al, 2005), and a NUP98-HOXA9 chimaera is generated in t(7;11)-AML (Nakamura et al, 1996). Nevertheless, the average level of HOXA9 transcripts in G1 and G3 was as low as 10% of that seen in other groups (evidenced by quantification of the results in Fig 4). Thus, an interesting possibility is that a pathway mediated by HOXA9 is not necessary or may even be antagonistic to the molecular mechanisms involved in t(8;21)- and inv(16)-AML. Low expression of CBFβ in G3 but not G1 may mean that the CBFβ-MYH11 protein negatively autoregulates expression of the wild type CBFβ allele.
In conclusion, we extracted and categorised t(8;21)/inv(16)-associated genes as t(8;21)-specific, inv(16)-specific, and genes common to both. This categorisation was enabled by a unique approach to gene extraction. While the presence of specific groups of selected genes correlated with the bimodular structures of the chimaeric proteins, it was notable t(8;21)- and inv(16)-AML display a significant degree of overlap in their gene expression signatures.
This work was supported by the programme for Promotion of Fundamental Studies in Health Sciences of the Pharmaceuticals and Medical Devices Agency (PMDA), by a Grant-in-Aid for Third Term Comprehensive Control Research for Cancer from the Ministry of Health, Labour and Welfare; and by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We are grateful to Ms. Sachiyo Mitani for technical assistance and Ms. Michika Kuji for secretarial assistance. M.S. is a participant in the 21st century COE program ‘Center for Innovative Therapeutic Development toward the Conquest of Signal Transduction Diseases’ at Tohoku University.