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NEOPLASIA
From the Department of Haematology, Cancer Research
Centre, Queen's University, Belfast, Northern Ireland; the Division of
Medical and Molecular Genetics, Guy's, King's & St Thomas'
School of Medicine, London; Department of Experimental
Haematology and Medicine, St Bartholomew's; and The Royal London
Hospital, London, United Kingdom.
Acute promyelocytic leukemia (APL) is associated with a reciprocal
and balanced translocation involving the retinoic acid receptor- Acute promyelocytic leukemia (APL) is a subgroup of
acute myelogenous leukemia (AML) typified by the t(15;17) leading to
fusion of the putative growth suppressor PML to the retinoic acid
receptor HOX genes encode a subset of homeodomain-containing
proteins, thought to bind DNA, which act as major regulators of
embryogenesis and organogenesis.6 HOX genes
contain a highly conserved 183-base pair (bp) region termed the
homeobox.7-9 The genomes of all animals analyzed to date
exhibit a distinct clustering of the homeobox genes, which are assumed
to have arisen by duplication and divergence from a primordial
HOX gene. In nonvertebrates one cluster exists HOX genes play a significant role in both normal and
dysregulated hematopoiesis.12-15 In rare cases of AML
involving a t(7;11)(p15;p15) or t(2;11)(q31;p15) translocation, the
HOXA9 or HOXD13 genes, respectively, are fused
with the NUP98 nucleoporin gene, underlying the disease
state.16-20 Furthermore, HOXA9 showed strong
correlation with AML and treatment failure when an oligo expression
array containing 6817 genes was used to segregate AML and acute
lymphoblastic leukemia (ALL) patients.21 The product of
the mixed lineage leukemia gene (MLL) is a positive
regulator for the maintenance of cell-specific HOX
expression via chromatin remodeling.22,23 MLL
has been implicated in fusions with more than 25 other genes in both
ALL (eg, MLL-AF4) and AML (eg,
MLL-AF9).24,25 Thus, alterations in
MLL may also disrupt HOX gene expression patterns in hematopoietic progenitor cells and contribute to a leukemic state.26
We have developed and validated a small-array
real-time quantitative polymerase chain
reaction (PCR) approach for the simultaneous and rapid measurement of
expression of 27 individual HOX genes (SMART-HOX). TaqMan probe-based chemistry (ABI Prism 7700;
Applied Biosystems, Foster City, CA) was used to attain high levels of specificity, and application of SMART-HOX to 5 leukemic cell
lines revealed novel patterns of gene expression. The aim of this study was to identify interactive HOX gene pathways in the
well-defined APL subgroup.
Leukemic cell lines and patient samples
RNA isolation, DNase treatment, and cDNA preparation
Real-time quantitative PCR (RQ-PCR) RQ-PCR was carried out using TaqMan probe-based chemistry (Applied Biosystems). This chemistry provides for a high level of specificity through the design of complementary oligonucleotide primers and 5'-reporter/3' quencher fluorogenic probes. The 5'-reporters for the HOX genes and endogenous controls (18SrRNA and GAPDH) were 6-carboxyfluorescein (FAM) and VIC (Applied Biosystems), respectively. In all cases the 3' quencher was 6-carboxy-tetramethylrhodamine (TAMRA). During the normal PCR process the fluorogenic probe is cleaved by the native 5'-exonuclease activity of Taq polymerase30 that releases the reporter from the quencher, resulting in the generation of a sequence-specific signal. Each additional cycle results in the release of reporter molecules from the respective probes. The fluorescence intensity is related to the initial number of RNA copies, which can be assessed by determining the threshold cycle (CT).31 All HOX-specific primers and probes were designed against GenBank-published sequences in association with Primer Express (Applied Biosystems). Endogenous controls were purchased as RNA-specific Pre Developed Assay Reagents (PDARs; Applied Biosystems). The universality of the Taqman system arising from the intrinsic constraints of Primer Express was exploited to measure simultaneously the expression of 27 HOX genes in singleplex mode. Control reactions lacking cDNA template, included to assess specificity, showed no appreciable amplification (CT more than 40; data not shown). The amplification reactions (12.5 µL) contained 50 ng cDNA equivalents (or control), 1 × Taqman universal PCR master mix, final concentrations of 5 mM MgCl2, 0.2 mM deoxyadenosine deoxycytosine deoxyguanosine triphosphate (dA/dC/dGTP), 0.4 mM deoxyuridine triphosphate (dUTP), 0.125 U AmpliTaq Gold, 2 µM primers (forward and reverse), and 200 nM TaqMan probe.Amplifications were performed following an initial 2-minute incubation at 50°C to allow uracil-N-glycosylase (UNG) to destroy any contaminating RNA, followed by treatment at 95°C for 10 minutes to inactivate the UNG enzyme and activate the AmpliTaq Gold DNA polymerase. This was followed by 40 to 45 cycles of denaturing at 95°C for 15 seconds and annealing/extension at 60°C for 1 minute. An ABI Prism 7700 Sequence Detection System equipped with a 96-well thermal cycler was used for the amplifications. Data were collected and analyzed with Sequence Detector v1.6.3 software (Applied Biosystems). To reduce the possibility of altered reaction kinetics contributing to
the difference in the PCR profile and hence the CT values,
the starting template concentration must be similar for each patient
and normal sample. For this reason, samples that gave an 18S
CT value outside the range of 10 ± 3 were excluded from
further analysis. This range of template concentration is well within
the guidelines of 5 orders of magnitude as set out in Applied
Biosystems' user bulletin no. 2. Relative quantitative (Q-PCR) data based on the Cloning, sequencing, and validation of RQ-PCR amplicons Oligonucleotides were designed and developed in association with Applied Biosystems. Due to the high degree of sequence homology between and among HOX paralog groups, it was essential to validate that each amplicon generated during the RQ-PCR process was the desired target by sequencing the product. PCR reactions using the gene-specific forward and reverse primers in the absence of the fluorogenic oligonucleotide probe were performed on the ABI Prism 7700 under exactly the same conditions as the RQ-PCR reactions. cDNA from several cell lines, a fetal brain library, and normal bone marrow were "pooled" to provide a source of template that would contain all potential HOX targets. Amplicons were identified by electrophoresis through a 2.5% agarose gel, excised, and purified using a CONCERT Rapid Gel Extraction System (Life Technologies) following the manufacturer's protocol. Purified amplicons were then directly cloned into the TOPO TA Cloning kit (InVitrogen, Paisley, United Kingdom) and transformed into TOP10F cells (InVitrogen). Plasmid DNA was extracted from ampicillin-resistant Luria-Bertani (LB) broth cultures by the CONCERT Rapid Plasmid Miniprep System (Life Technologies) and sequenced using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems) along with M13F (17 mer) or M13R (21 mer) primers with an ABI 310 sequence detection system (Applied Biosystems). At least 5 individual colonies were sequenced for each cloning reaction. All sequences were searched against GenBank and Swiss Protein Database (SWISSPROT) using Basic Logic Alignment Search Tool (BLAST)33 and fast all protein or nucleotide comparison search tool (FASTA)34 and verified as being specific for the original target gene.Differentiation of NB4 cells with ATRA treatment assays NB4 cells were seeded at a concentration of 2 × 104/mL in standard T-75 tissue culture flasks. Cells were either untreated (control) or treated with 1 µM ATRA or an equivalent volume of vehicle (EtOH) for the indicated times. Cell proliferation was monitored by the direct count method of viable cells using standard Trypan Blue exclusion assays. NB4 cell differentiation was monitored using a direct immunofluorescence assay measuring the myeloid cell surface molecule CD11b as a marker. The phycoerythrin (PE)-conjugated monoclonal anti-CD11b clone 2LPM19c (Dako A/S Denmark) antibody was used in all experiments with appropriate isotype controls (IgG1). The percentage of CD11b+ cells was calculated by the EXPO32 analyzer software (Applied Cytometry Systems, Sheffield, United Kingdom). Morphologic assays were carried out on cytospun specimens using May-Grünwald Giemsa staining and light microscopy.
Design, development, and validation of the RQ-PCR oligonucleotides used The primers and probes used in the reactions (Table 1) were designed and developed using the latest available GenBank sequence information and Primer Express. Direct DNA sequencing of at least 5 clones per target (data not shown) validated the specificity of the amplicons. The amplicon lengths ranged from 56 bp to 107 bp (Table 1). Twenty-seven HOX targets were successfully detected by the designed assays. Plasmid DNA from clones that were sequenced showed either the original target or self-ligation of the cloning vector.
SMART-HOX profiling in leukemic cell lines Initially the SMART-HOX technology was applied to several well-characterized leukemic cell lines (Figure 1). The objective was to assess the expression of 27 HOX genes in various leukemic cell lines in a comprehensive and quantitative manner. Previous studies on leukemic cells have involved the use of standard PCR, Northern blot, or RNase protection assays, which have limitations either due to the amount of template required or the degree of quantitation obtained.15,35-39 SMART-HOX has evolved the measurement of relative gene dosage differences using specific, validated TaqMan reagents. Figure 1 depicts a 2-dimensional readout of the CT values obtained for each cell line (columns) for each particular HOX gene (rows) corrected for starting template concentration by the 18S endogenous control. The values obtained were also validated using GAPDH controls, which duplicated the findings in each of the cell lines (data not shown). The CT value for GAPDH in the KG1a cell line was markedly higher than the other cell lines (data not shown), while the 18SrRNA values were consistent among the samples. The CT value reflects the initial concentration of the target cDNA in that it is inversely proportional to the target concentration. Because the CT value is obtained during the exponential phase of the PCR reaction, a decrease of 1 CT value between comparative samples is equivalent to a doubling of the initial target concentration that is, gene expression
= 2( CT). The lower the CT value,
the higher the expression of that particular HOX gene. A
color-coded system is used to highlight differences in HOX
gene expression among the leukemic cell lines tested. The highest
expressed HOX genes (lowest CT values 20 to 23)
are colored red, while very low/absent expression (CT
values more than 38) are colored gray. The results (Figure 1)
were consistent with and expanded on previous studies. There was a
preponderance of HOXB expression in the erythroid cell line
HEL,27,39 whereas the more myeloid-type cell lines (eg,
U937) tended to express the HOXA genes.40 The
general high level expression of some 66% of the subset of
HOX genes studied was noted in the NB4 cell line. Certain
HOX genes, particularly A7, A10, and
C9, appear to be highly expressed ubiquitously throughout
the cell lines studied. These HOX genes may play a critical
role in basal cellular processes independent of the lineage subtype or
differentiation state. In some cases the level of expression of
particular HOX genes, A9 in KG1a cells and
D13 in NB4 cells, approached the level observed for
GAPDH (CT 18 to 20) for the same cells. This was a surprising result because these transcription factors are usually expressed at low levelshence the use of PCR and RNase protection assays in the past to observe their occurrence. Additional novel findings were in the relatively low expression of most of the HOX genes studied in HL60 cells (only 30% scored as
moderately expressed or higher) and in the similarity of profile
between HL60 and U937 cells. The high expression of HOXD13
in the HL60, NB4, and HEL cell lines was unexpected, because
D13 was originally considered to be a gene involved in limb
development,41,42 although it was recently identified as a
NUP98 fusion partner in some AML cases.16,19,20
The significance of some of these findings and the link between
HOX gene expression and potential upstream regulators show
the strength of investigating an array of HOX genes at the
same time in a quantitative manner. This has previously been difficult
and time-consuming. Insights into the role of certain HOX
genes in leukemogenesis are suggested from this approach.
Altered HOX gene expression in differentiating NB4 cells ATRA treatment of NB4 cells over a 96-hour period resulted in a significant decrease in the rate of cell proliferation (Figure 2A) as well as the induction of a differentiation response, observed as an increased expression of the cell surface marker CD11b (Figure 2B). Morphologic changes, in particular the presence of band neutrophils, were observed following 96-hour ATRA treatment of NB4 cells but were not seen in vehicle-treated cells (Figure 2C-D). Morphologic changes occurred at the same stage as an increase in apoptosis was observed (data not shown). Taken together these results confirmed the ability of the NB4 cells used to differentiate in the presence of ATRA. To gain functional insights into this differentiation process, the expression of 27 HOX genes was monitored at 2 different time points, one following short-term (4 hours) exposure to ATRA (Figure 3A), the other (48 hours) following the establishment of the differentiation response (Figure 3B). The levels of HOX gene expression following terminal differentiation were not investigated due to the high level of apoptosis in these cells. HOX gene expression (as actual CT values corrected for 18S) was compared with vehicle. Most HOX genes showed no significant difference in expression 4 hours following ATRA treatment (Figure 3A and data not shown). However, there was a moderate increase in the expression of several HOXA cluster genes (decrease in CT values) and a moderate decrease (increase in CT values) in the expression of other genes (eg, HOXB9). Following differentiation (48-hour time point) there appeared to be a moderate down-regulation (increased CT values) for many of the HOX genes studied. The fold difference in HOX gene expression was calculated from the following formula: fold difference = 2CT, where
 CT = CT (EtOH)  CT
(ATRA). The expression of HOXC12 increased 20-fold within 4 hours and was maintained or slightly enhanced (23-fold) following
longer-term (48 hours) ATRA treatment (Figure 3B-C). The constitutively
highly expressed HOXD13 was down-regulated 5-fold within 4 hours of ATRA treatment that was also maintained (6.5-fold) during the
differentiation process. These significant changes occurred in
the presence of more moderate differences in several other genes,
including HOXA1, HOXA11, and HOXB9, suggesting a
degree of specificity in the response to ATRA rather than a global
effect on HOX gene expression.
SMART-HOX profiling in APL patient samples The SMART-HOX platform was applied to cDNAs obtained from 16 PML-RAR + cases of APL and compared
with normal bone marrow cDNA samples (n = 3). SMART-HOX
assays were carried out in triplicate on a 96-well plate format with
endogenous control genes (18SrRNA and GAPDH) and
analyzed with corresponding negative controls (minus template).
CT values were normalized to either 18S or
GAPDH values, which gave comparable results. 18S
normalized values were plotted (Figure
4). APL patients exhibited a
global down-regulation (increase in CT value) of
HOX gene expression, within paralogs and across clusters for
all 26 genes assayed (HOXC5 was omitted due to insufficient quantity), compared with healthy controls (Figure 4).
The degree of down-regulation did not appear to follow any defined
pattern. The most significant fold reductions in expression (Figure
5) included such divergent genes
as HOXA4, HOXA11, HOXB3, HOXC9, and HOXD8. Of the
26 genes assayed, the most significant changes occurred in 71% of the
HOXA and HOXC clusters (5 of 7), 37.5% of the
HOXB cluster (3 of 8), and 60% of the HOXD
cluster (3 of 5). The fold decrease in expression ranged from 1.8 (HOXC4) to 7.4 (HOXA4). A pattern of expression
throughout the HOXA cluster whereby the low (1 to 4) and
high (10 to 11) numbered paralogs were expressed at lower levels than
the mid (5 to 9) paralogs was observed in both the healthy and APL
patient samples. This pattern of expression has also been noted in
other AML and ALL patient samples (data not shown).
The PML-RAR Leukemic cell lines provide important models of dysregulated
hematopoiesis, but immortalization or transformation can directly perturb the expression of genes involved in regulatory processes, such
as HOX genes. It is possible that this holds for the NB4 cell line, which showed moderate to high expression of 18 of the 27 genes assayed. NB4 cells were originally isolated from long-term cultures of APL leukemic blasts as a late event and have a complex karyotype.49 The cells do, however, retain the
PML-RAR ATRA treatment of NB4 cells resulted in moderate up-regulation of HOXA1 (4- to 5-fold) as previously reported50 and down-regulation of several other genes, namely HOXA9, A11, B9, C5, and C9 (Figure 3A-B). A more significant up-regulation of HOXC12 (at least 20-fold) and down-regulation of HOXD13 (at least 5-fold) were also observed (Figure 3C). In both cases the ATRA effect occurred within 4 hours of treatment and was increased at 48 hours when the NB4 cells had begun to differentiate. These results agree with reports that pharmacologic doses of ATRA release the block in myeloid differentiation and suggest that early and sustained modulation of particular HOX genes is involved in this process. To examine the relationship between HOX genes and APL in
vivo, diagnostic PML-RAR
We thank Dr Steve Picton and Dr Adam Corner (Applied Biosystems) for their expert advice and support, Dr Colin McGuckin and Nico Forraz for providing normal bone marrow samples, and Dr Francisco Lopez-Gordillo for his technical assistance.
Submitted November 6, 2001; accepted October 15, 2002.
Supported by the Northern Ireland Leukaemia Research Fund (A.T., C.M.O.), Leukaemia Research Fund of Great Britain (F.C., D.G.), and Action Cancer.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Terence R. J. Lappin, Queen's University Belfast, Department of Haematology, University Floor, Tower Block Belfast City Hospital, Lisburn Rd, Belfast BT97AB, Northern Ireland; e-mail: t.lappin{at}qub.ac.uk.
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© 2003 by The American Society of Hematology.
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  X. Zhang, Z. Lian, C. Padden, M. B. Gerstein, J. Rozowsky, M. Snyder, T. R. Gingeras, P. Kapranov, S. M. Weissman, and P. E. Newburger A myelopoiesis-associated regulatory intergenic noncoding RNA transcript within the human HOXA cluster Blood, March 12, 2009; 113(11): 2526 - 2534. [Abstract] [Full Text] [PDF]
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L. McCallum, S. Price, N. Planque, B. Perbal, A. Pierce, A. D. Whetton, and A. E. Irvine A novel mechanism for BCR-ABL action: stimulated secretion of CCN3 is involved in growth and differentiation regulation Blood, September 1, 2006; 108(5): 1716 - 1723. [Abstract] [Full Text] [PDF]
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A. Mamo, J. Krosl, E. Kroon, J. Bijl, A. Thompson, N. Mayotte, S. Girard, R. Bisaillon, N. Beslu, M. Featherstone, et al. Molecular dissection of Meis1 reveals 2 domains required for leukemia induction and a key role for Hoxa gene activation Blood, July 15, 2006; 108(2): 622 - 629. [Abstract] [Full Text] [PDF]
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C. S. Wilson, G. S. Davidson, S. B. Martin, E. Andries, J. Potter, R. Harvey, K. Ar, Y. Xu, K. J. Kopecky, D. P. Ankerst, et al. Gene expression profiling of adult acute myeloid leukemia identifies novel biologic clusters for risk classification and outcome prediction Blood, July 15, 2006; 108(2): 685 - 696. [Abstract] [Full Text] [PDF]
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J. Bijl, A. Thompson, R. Ramirez-Solis, J. Krosl, D. G. Grier, H. J. Lawrence, and G. Sauvageau Analysis of HSC activity and compensatory Hox gene expression profile in Hoxb cluster mutant fetal liver cells Blood, July 1, 2006; 108(1): 116 - 122. [Abstract] [Full Text] [PDF]
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+ acute promyelocytic leukemia identified by
small-array real-time PCR
Top
Abstract
Introduction
, respectively. Reverse
transcription of the RNA pools was performed using murine Maloney
leukemia virus native reverse transcriptase (MMLV-RT) at a
concentration of 105 U/mL in the presence of 0.5 mM
deoxyribonucleoside triphosphate (dNTP), 5 × 103
U/mL RNase inhibitor, and 50 µM random hexamer primers (all from Life
Technologies) at 42°C for 2 hours (RT+). The
RT
,
4-hour EtOH; 
, 4-hour ATRA) or (B) the onset of
differentiation after ATRA exposure (
