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a Department of Medicine, Veterans Affairs Medical Center, University of California at San Francisco, California, USA;
b Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada;
c Department of Medicine and Comprehensive Cancer Center, University of California at San Francisco, California, USA
Key Words. Hematopoiesis • Hematopoietic stem cells (HSCs) • Homeobox genes • Microarray • Gene expression profiling
Correspondence: H. Jeffrey Lawrence, M.D., Department of Medicine, Hematology Research (151H), Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121, USA. Telephone: 415-221-4810, ext. 3340; Fax: 415-750-6959; e-mail: jeffl{at}medicine.ucsf.edu
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ABSTRACT |
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INTRODUCTION |
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The function of HOX genes in normal murine hematopoiesis has been explored both in knockout mice and in murine models of retrovirally driven overexpression. HOXA9 has an important role in normal hematopoiesis, as HOXA9-deficient mice have a variety of myeloid and lymphoid defects, as well as abnormalities in hematopoietic stem cell (HSC) function [4, 5]. By contrast, no consistent hematopoietic abnormalities have been described in mice lacking the HOXA10 gene. These findings suggest that, although both HOXA9 and HOXA10 are expressed in primitive hematopoietic cells, the two genes have distinct functions during normal blood cell differentiation. In accord with this notion is the fact that HOXA9 and HOXA10 proteins have little homology to one another outside of the 60-amino-acid DNA-binding homeodomain (HD).
However, overexpression of either HOXA9 or HOXA10 in murine marrow cells produces similar blood cell abnormalities, including a marked expansion of HSCs and committed progenitors with eventual transformation to acute myeloid leukemia (AML) [6–8]. These similar biologic effects on blood cell development suggest that HOXA9 and HOXA10 modulate common genetic programs in blood cells when they are over-expressed. Moreover, in recent studies, we have documented striking overlap in the functional effects of NUP98-AbdB class HOX fusions, as tested with naturally occurring leukogenic fusion such as NUP98-HOXD13 and engineered fusions such as NUP98-HOXA10 [9]. Such findings further suggest a high degree of target gene overlap, at least for HOX within the AbdB class of HOX (paralogs 9–13).
Since homeobox genes encode DNA-binding proteins, it has been presumed that HOX proteins function as transcription factors. Recent papers have described the transcriptional profiles of certain HD proteins using microarray and real-time reverse transcription polymerase chain reaction (RT-PCR) technologies in an effort to identify potential gene targets and their associated downstream molecular pathways. These studies have examined the genes modulated by the expression of HOXC13, HOXD3, the non-HOX HD proteins, PAX6, and the fly HOX1 homologue, labial, in nonhematopoietic tissues [10–13]. Another study examined the expression profile of the leukemogenic fusion gene, NUP98-HOXA9, in an immortalized myeloid cell line [14], but no study published to date has attempted to identify genes modulated by a wild-type HOX protein in primary human hematopoietic cells.
In a recent paper, our laboratory published a description of the HOXA9 transcriptome in human leukemic cell lines, using a transient overexpression strategy in three cell lines, two myeloid and one lymphoid [15]. In that study we observed modulation of a large number of genes within 24 hours of introduction of a HOXA9 expression vector in these cells. The modulated genes represented a wide variety of functional groups, including oncogenes, cell-cycle proteins, enzymes, membrane proteins, and other transcription factors. Interestingly, a number of these genes are known to be part of the transcriptome of normal HSCs and to be similarly modulated in primary samples of human AML, suggesting that these genes are authentic biologic targets of HOXA9 [16].
Remarkably, in that study the gene-expression profiles observed for the myeloid and lymphoid lines were dramatically different, indicating that the transcriptional effects of HOXA9 are highly dependent on cell context. This observation raises questions as to whether the HOXA9 targets identified in aneuploid immortalized myeloid cell lines would match the gene targets for HOXA9 in normal hematopoietic cells. To answer this question, we have studied the expression profile of the HOXA9 protein in human umbilical cord CD34+ cells. In addition, we have used this same system to examine the transcriptome of the related HOXA10 protein, permitting a comparison of the genes modulated by these two closely related transcription factors.
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MATERIALS AND METHODS |
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Retrovirus Constructs and Virus Production
The MIG vector is a murine stem cell virus (MSCV)–based retroviral vector [17] that contains an internal ribosomal entry site (IRES) upstream of the green fluorescent protein (GFP) coding sequence [8]. The MIG-HOXA9 and MIG-HOXA10 vectors were made by cloning in HOXA9 or HOXA10 cDNAs upstream of the IRES-GFP region in the MIG plasmid. This vector design allows for coexpression of HOX and GFP proteins. High titer retrovirus (~1 million infectious viral particles per ml) was obtained by transfecting Phoenix amphotropic packaging cells (kindly provided by Gary Nolan, Stanford University, Stanford, CA) cultured in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum using the CaPO4 method (Cell Phect; Amersham Biosciences, Chandler, AZ, http://www.amersham.com) and harvesting the virus-containing medium (VCM) 36–60 hours later. After filtration through a 0.45-µm filter, the VCM was used without storing or freezing.
Cell Culture and Retroviral Transduction
Five separate CB units were analyzed in three separate experiments. Cord 1 was divided into two equal samples: one for HOXA9 transduction and one for HOXA10 transduction. Cords 2 and 3 were combined to form a single sample, Cord23mix, which was then segregated into two equal samples: one for HOXA9 transduction and one for HOXA10 transduction. Cords 4 and 5 were also combined to form a single sample, Cord45mix, for HOXA10 transduction for the study of later changes in gene expression.
On day 0, CD34+ CB cells were cultured at a concentration of 1 to 2 x 105 per ml and prestimulated by incubation in complete serum-free medium (SFM) consisting of Iscove’s modified Dulbecco’s medium with bovine serum albumin, insulin, transferrin (Stem Cell Technologies), 10–4 M ß-mercaptoethanol (Sigma-Aldrich Company, Oakville, ON, Canada, http://www.sigmaaldrich.com), along with five recombinant human growth factors, including 100 ng/ml Flt-3 ligand (Immunex, Seattle, http://immunex.com), 100 ng/ml Steel factor (Terry Fox Laboratory), 20 ng/ml interleukin-3 (IL-3; Novartis International, Basel, Switzerland, http://www.novartis.com), 20 ng/ml IL-6 (Cangene Corp., Mississuaga, ON, Canada, http://www.cangene.com), and 20 ng/ml G-CSF (Stem Cell Technologies).
On day 2, cells were resuspended in filtered VCM supplemented with the same five cytokines and 5 µg/ml polybrene (Sigma), then placed in fibronectin-coated dishes (Sigma) previously loaded with VCM for 1 hour. This infection protocol was repeated on day 3. Individual cord samples were transduced with MIG-control, MIG-HOXA9, or MIG-HOXA10 retroviral vectors. The transfection efficiencies, as measured by GFP expression, ranged from 16%–24%. On day 4, cells were transferred to fresh SFM with growth factors. On days 5 or 8, 3.9 to 10.8 x 104 viable (propidium iodide negative) CD34+ GFP+ CB cells were isolated by FACS. Cells were cultured in VCM media either for 3 days (HOXA9 or HOXA10 day 3) or for 6 days (HOXA10 day 6) prior to harvesting.
RNA Isolation and Amplification
Sorted cells were resuspended in Trizol reagent (Life Technologies, Rockville, MD, http://www.lifetech.com), and RNA was isolated according to the manufacturer’s instructions using glycogen as a coprecipitant. RNA from MIG-control, MIG-HOXA9, or MIG-HOXA10 was amplified by two rounds of in vitro transcription, as previously described [15, 18]. RNA from 10,000 to 40,000 cells was amplified to 12–27 µg of amplified RNA (aRNA).
cDNA Probe Preparation and Hybridization to cDNA Microarrays
The 1.5µg of a RNA from MIG-control–transduced CB cells were labeled with Cyanine 3 (Cy3), and 1.5 µg of aRNA from MIG-HOXA9- or MIG-HOXA10-transduced CB cells were labeled with Cyanine 5 (Cy5). MIG-control was combined with either MIG-HOXA9 or MIG-HOXA10 samples and hybridized to two cDNA microarrays representing a 42,000 human cDNA clone set (~20,000 genes per slide) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com, with additional customized cDNAs printed by the Haqq laboratory). The microarrays were printed with cDNA clones onto poly-3-lysine–coated glass slides and postprocessed as described at http://www.microarrays.org/pdfs/PostProcessing2001.pdf. Samples were hybridized to the micro-arrays overnight in a 63°C water bath and were washed twice with 2x sodium chloride/sodium citrate (SSC) with 0.01% sodium dodecyl sulfate (SDS) and once with 0.5x SSC. The Axon 4000B scanner and GenePix 3.0 software (Axon Instruments/Molecular Devices Corp., Union City, CA, http://www.moleculardevice.com) were used to collect and perform initial analysis of Cy3 and Cy5 fluorescent images. Obvious array or hybridization artifacts were flagged manually and excluded from further analysis.
Microarray Samples and Data Analysis
Microarray analysis was performed in triplicate for each condition tested. Cord 1 and Cord23mix were biological replicates, making up two of the arrays. An additional array of one or the other of these samples was run as a technical replicate, comprising the third of the arrays for analysis of HOXA9 and HOXA10 day 3–transduced cells. Cord45mix was run in triplicate as technical replicates for HOXA10 day 6–transduced cells. GenePix 3.0 result files were uploaded and maintained in the NOMAD database, as previously described [15]. Data were analyzed using downloadable significance analysis of microarrays (SAM), Eisen Cluster, TreeView, and GenMAPP programs available through the following Websites: http://www-stat.stanford.edu/~tibs/SAM/, http://rana.lbl.gov/EisenSoftware.htm, and http://www.genmapp.org.
Statistical Analysis
SAM, a modified t-test algorithm, was used to identify genes with statistically significant changes in expression (see http://www.utulsa.edu/microarray/Articles/sam%20manual.pdf) [19]. The dataset was filtered to exclude genes in which more than 20% of measurements were flagged. SAM was first run in one-class response mode to determine genes in common among HOXA9 and HOXA10 cells, and the statistical delta parameter was chosen to result in a predicted false discovery rate (FDR) of 1%. Missing data were replaced by row-average imputation. Differential expression patterns comparing HOXA9 and HOXA10 were determined using the two-class (unpaired) t-test in the SAM package.
Individual microarray data files were normalized in NOMAD and grouped. Grouped files were then analyzed in Eisen Cluster for log2 transformation, and hierarchical-average linkage clustering. Only those genes with induction or repression in expression greater than twofold for four or more samples were accepted for further analysis. The resulting data were then used for visualization of clusters in TreeView.
Quantitative Real-Time PCR
Raw data were obtained on an ABI Prism 7900HT SDS machine (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) using fluorogenic SYBR Green I double-stranded DNA-binding dye chemistry during real-time RT-PCR [20]. Briefly, 0.5µg of aRNA was reverse transcribed to cDNA using the Superscript II polymerase (Invitrogen). For analysis, approximately 250 ng of cDNA was mixed with 125 nM forward and reverse primers, water, and 2x SYBR Green I master mix to a total volume of 20 µ1 and amplified by 40 PCR cycles. Each run included a dissociation curve to screen for nonspecific products. To adjust for variations in loading, the Ct values for each gene were normalized against the Ct values for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase, (G3PDH). Relative gene expression was calculated using the 2–
Ct method [21]. All oligos were purchased from Operon (Alameda, CA, http://www.operon.com).
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RESULTS |
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shows a schema of the experimental design). Real-time RT-PCR of RNA from day-3 samples revealed that a six- to sevenfold increase in mRNA levels of HOXA9 and HOXA10 (Fig. 2A) was achieved in the transduced cells. CB mRNA from HOXA9- and HOXA10-transduced samples were compared with MIG only–transduced controls through hybridization to cDNA microarrays to provide a differential gene expression profile. These microarray data were then subjected to stepwise statistical analysis.
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). Of the approximately 42,000 cDNA array clones, 1,998 genes were similarly modulated by both HOXA9 and HOXA10, as compared with the MIG control. By setting the value at 0.614, the FDR was 0.87%, and only 17 out of 1,998 genes were predicted to be false. Thus, SAMs show that these related HOX proteins are able to modulate many of the same genes. The small number of differentially modulated genes (i.e., upregulated by HOXA9 and downregulated by HOXA10, or the reverse) was represented almost entirely by expressed sequence tags of unknown function.
In a second step, Eisen Cluster analysis was performed, introducing threshold values to increase the likelihood of the identified genes being truly regulated and of biological significance. (Fig. 2B). Triplicate microarray data from day 3–harvested HOXA9- or HOXA10-transduced samples were filtered by the Eisen Cluster analysis program for genes, with four out of six observations showing twofold induction or repression. Using these more stringent criteria, 115 genes were modulated by one or both of the over-expressed genes. The gene expression profiles for HOXA9 and HOXA10 are shown in Figure 2C, broken down into four groups (i–iv). Group i consists of genes that are similarly regulated by HOXA9 and HOXA10. Groups ii and iii show genes that are differentially modulated by HOXA9 and HOXA10—for example, upregulated by HOXA9 yet downregulated by HOXA10. The largest group, group iv, represents genes that are repressed by both HOXA9 and HOXA10. This Eisen Cluster analysis suggests that there are many more genes regulated in a similar fashion by HOXA9 and HOXA10 than are differentially regulated.
Annotated genes that passed the Eisen Cluster filter for at least twofold differential expression, which were similarly regulated in more than one cord, and which were also determined significant by SAM, are presented in Tables 1 and 2. Table 1 is a list of 35 named genes that were either induced or repressed by HOXA9 and were also modulated by HOXA10. Table 2 lists genes that were modulated by HOXA10 after 3 days and remained modulated or were not modulated until 6 days of overexpression. While several genes were shown to be modulated at both time points, a larger number of genes was identified in the day-6 sample, which probably reflects the progressive modulation of secondary and tertiary genes over time. Several mRNAs that were previously detected in CD34+ cells are noted in both tables, showing that both HOX proteins modulate a number of genes that are active in HSCs [16].
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). The amplitude of the difference in gene expression as measured by real-time RT-PCR was often greater than that observed by microarray analysis, though results were always concordant in the direction of change.
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Activation of the Wnt Signaling Pathway by HOXA9 and HOXA10
HOXA9 and HOXA10 were found to positively regulate genes in the Wnt signaling pathway (Fig. 4). Wnt10B demonstrated a 4.5-fold increase in HOXA9-transduced cells, with a 3.1-fold increase in HOXA10-transduced cells, and this upregulation was validated by real-time PCR. In addition, two Wnt receptors, Frizzled 1 and Frizzled 5, were also upregulated, with Frizzled 1 showing approximately twofold upregulation by both HOXA9 and HOXA10, and Frizzled 5 showing two- to fourfold upregulation by HOXA10; these results were also validated by real-time PCR. The ability of these two HOX proteins to activate Wnt and Frizzled genes, given the key role of this pathway in stem cell self-renewal, may explain, at least in part, the biologic effects of HOX proteins on primitive hematopoietic cells.
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and Tables 1 and 2 show that many genes are downregulated by both HOX proteins, as was seen in our previous expression profile study in hematopoietic cell lines. Among the downregulated genes in this survey is CYBB, a respiratory burst oxidase component pg91(phox), which is known to be expressed in maturing myeloid cells and has previously been shown to be repressed by HOXA10 (Table 2) [24]. Other significantly downregulated genes included Tip60 and vimentin. Vimentin is an intermediate filament protein that, in blood cells, increases with monocytic differentiation. Support for the notion that the vimentin gene could be a target of HOXA proteins comes from the observation that its expression is lost in ovarian tumors that overexpress HOXA7 [25]. Furthermore, the vimentin promoter and enhancer region has been shown to have binding sites for HOX proteins [26]. Thus, at least two genes associated with myeloid differentiation are inhibited by HOXA9 and/or HOXA10. Tip60 is a 60-kDa Tat– interactive protein that normally represses STAT3 and is involved in sensing DNA damage and inducing apoptosis [27, 28]. Thus down-regulation of Tip60 could be associated with improved survival and increased genomic instability, thereby predisposing to malignant transformation.
Inhibition of the Erythroid Differentiation Program by HOXA9 and HOXA10
To identify other molecular pathways affected by HOXA9 and HOXA10, these microarray data were also analyzed with the Gen-MAPP program designed by the Gladstone Institute in affiliation with the University of California at San Francisco (http://www.genmapp.org/download.asp). This analysis demonstrated that HOXA9 (1) and HOXA10 (3) inhibit enzymes of the heme bio-synthetic pathway. Likewise, HOXA9 (4) and HOXA10 (2) also inhibit globin genes (Fig. 5). Not included in the figure is inhibition of the pathway rate-limiting enzyme, ALAS-2 by HOXA10 (Table 2). SAM showed that HOXA9 and HOXA10 also down-regulate globin genes expressed from the ß-globin and 
-globin loci (Tables 1 and 2; Fig. 5). Thus, both HOXA9 and HOXA10 appear to effect a general repression of erythroid-specific genes, and this is consistent with previous observations [29].
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DISCUSSION |
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With the above caveats in mind, the similarity in gene-expression profiles is consistent with the hematological phenotypes seen in mice transplanted with either HOXA9-or HOXA10-transduced marrow cells. In both cases, recipient animals display a marked increase in stem cell numbers, colony-forming unit-spleen, and clonogenic progenitors with eventual transformation to AML. Similar blood cell abnormalities have been reported in mice transplanted with marrow cells transduced with more distantly related HOX genes, including HOXB3 and HOXB6. An exception to this general phenomenon is the case of HOXB4, whose moderate overexpression does not interfere with hematopoietic differentiation or lead to AML but still expands the HSC population. A subtle difference between the phenotypes associated with HOXA9 and HOXA10 overexpression in murine marrow is an increase in megakaryoblastic-like colonies in HOXA10-transduced cells [6]. However, we compared our dataset of modulated genes with two previous databases, one for megakaryocytic-specific genes and one for platelet-specific genes, and found no genes in common [30, 31].
Although there have been no reports of any consistent hematopoietic abnormalities in HOXA10–/– mice, HOXA9–/– mice display an array of hematological defects, including lower numbers of mature granulocytes and lymphocytes, a blunted response to G-CSF, small spleens, lower numbers of myeloid and B-lymphoid progenitors, aberrant fetal thymocyte development, and reduced repopulating ability in competitive transplantation assays [4, 5]. This biologic difference between HOXA9 and HOXA10 may be explained by a recent study in which the expression of HOXA, B, C, and D changes were measured in Lin– Sca+ fetal liver blood cells using quantitative RT-PCR. In that study, HOXA9 was one of the most strongly expressed HOX genes in primitive hematopoietic cells, whereas HOXA10 was expressed at much lower levels [32].
Although the study reported here and in our previously published survey in leukemic cell lines was performed with the same microarray gene set, following similar protocols, there were differences in the profiles of genes modulated by HOXA9. This difference could be due, in part, to technical differences between the two studies. Different transfection strategies were used in the two studies, perhaps resulting in different expression levels of the transduced gene. RNA in this study was harvested from the CD34+ cells 3–6 days after transduction, whereas in the prior study RNA was harvested after only 24 hours post-transduction. However, it seems likely that the most important difference is the target cell used. The myeloid cell lines used in the prior study, K562 and U937, are aneuploid, factor-independent immortalized cell lines derived from leukemic samples, which harbor a variety of mutations and alterations in chromatin structure. All of the above factors could modify their response to an introduced transcription factor. It may be that a number of genes modified in the current study were not detected in the previous study due to the possibility that they were already fully activated in the leukemic cell lines.
HOXA9 and HOXA10 appear to repressseveral genes involved in erythroid differentiation, including globin genes and several genes of the heme synthetic pathway. Prior studies have suggested that HOX genes could inhibit red cell maturation; that is, mice transplanted with marrow cells overexpressing either HOXA9 or HOXA10 are anemic [6, 27, 33]. Another HOXA cluster member, HOXA5, has also been shown to suppress erythroid differentiation when overexpressed in human CD34+CD38– cells [34]. Similar results have been seen with overexpression of HOXB6, which suppresses hemoglobinization in both cell lines and primary bone marrow cells [35, 33]. Recipients of HOXB6-transduced marrow are anemic and have lower numbers of splenic BFU-E [33]. Conversely, HOXB6-deficient mice have higher numbers of adult and fetal BFU-E [36]. There is evidence that HOX proteins can bind to various sites within the ß-globin locus, including the locus control region, the 
-globin promoter, and the A-globin enhancer, perhaps thereby directly repressing erythroid genes [37].
Of the genes upregulated by HOXA9 and HOXA10 determined by microarray analysis, seven were validated by QRT-PCR. Six of these genes have been reported to be expressed in HSCs or are thought to have a role in cell proliferation and self-renewal. Two of these genes, ALDH1 and Iroquois 3 (IRX3) were noted to be upregulated by HOXA9 in myeloid cell lines in our previous survey [15].
IRX3, a member of the 6-member IRX/TALE family of homeobox genes, plays an important role in the early development of the nervous system [38]. Two prior studies have suggested that HOX proteins may regulate the expression of IRX genes. In Xenopus, IRX5 appears to be a direct gene target for HOXB4 [23]. In addition, HOXA3 has been shown to repress IRX3 expression during hindbrain tissue patterning and the formation of motor neurons in the chick [39]. Until now, there has been no published evidence directly linking Iroquois genes to hematopoiesis, although Fused toes (Ft) mutation in mice has been shown to be a deletion, including IRX3 and two other neighboring Iroquois family members [40]. These animals suffer a variety of skeletal abnormalities reminiscent of HOX mutations, and heterozygotes have thymic hyperplasia [41, 42].
ERG, or Ets-related gene, is expressed in HSCs [16]. ERG modulates the developmental progression of thymocytes and B-cell maturation [43]. Maintained expression of ERG results in increased cell proliferation and self-renewal of myeloid progenitors, impaired B-cell differentiation, and arrested erythroid development [44]. These findings are similar to those observed in mice reconstituted with HOXA9- and HOXA10-expressing marrow cells.
Very long–chain acyl-CoA synthetase I (VLCS), regulates the production of long chain acyl-CoA esters, known to affect the regulation of signal transduction, lipid synthesis, energy metabolism, ion flux, and gene expression [45].
Although ALDH1 is expressed at significant levels in HSCs [46], its possible regulatory function in HSCs is still unknown, although it confers drug resistance to certain chemotherapeutic agents such as cyclophosphamide [47]. ALDH1 participates in the oxidation of retinal to all-trans retinoic acid [48], a molecule that exerts profound and complex effects on the proliferation and maturation of both HSCs and more committed progenitors [49–51]. Our data raise the novel possibility that HOXA9 and HOXA10 may be important in inducing the genes involved in retinoic acid synthesis in HSCs. This hypothesis is further bolstered by the observation that two other genes in this synthetic pathway, alcohol dehydrogenase 4 and retinal dehydrogenase homologue, were also upregulated by HOXA10, and like ALDH1, both have been identified as part of the CD34+ transcriptome [16].
Wnt10B is a member of the Wingless family of secreted glycoprotein signaling molecules. Wnt proteins and their Frizzled receptors appear to play an important role in normal hematopoiesis, in which they affect multiple processes, including HSC renewal, stem cell survival and proliferation, and commitment and cellular differentiation [52, 53]. Wnts play regulatory roles in multiple signaling pathways, including ß-catenin–TCF, c-Jun-N-terminal kinase (JNK), Ca2+-release, and cGMP regulation [54, 55]. Wnt10B is expressed at varying levels in T-cell, B-cell, and myeloid and erythroid hematopoietic cell lines [56]. Notably, Wnt10B enhanced colony formation of mixed or myeloid lineage progenitors and repressed formation of erythroid progenitors in CD34+ cells [56], an effect remarkably similar to that of HOXA9 and HOXA10.
In conclusion, this study suggests that HOX proteins participate in the transcriptional activation of a number of genes that regulate stem cell proliferation and self-renewal, and in the inhibition of differentiation by repressing the transcription of lineage- and maturation-related genes.
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ACKNOWLEDGMENTS |
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REFERENCES |
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-globin enhancer-binding protein is a product of the HOXB2 (HOX2H) locus. Blood 1994;83:1420–1427.This article has been cited by other articles:
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J.J. Kim, H.S. Taylor, Z. Lu, O. Ladhani, J.M. Hastings, K.S. Jackson, Y. Wu, S.W. Guo, and A.T. Fazleabas Altered expression of HOXA10 in endometriosis: potential role in decidualization Mol. Hum. Reprod., May 1, 2007; 13(5): 323 - 332. [Abstract] [Full Text] [PDF]
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